Atomic Force Microscope Manipulation of Living Cells

ABSTRACT

Techniques for atomic force microscope manipulation of living cells include functionalizing a nanoscale tip of a microscale cantilever with a first ligand for a first receptor associated with a surface of a first type of cell. The method further comprises controlling the cantilever to cause the first ligand on the nanoscale tip to contact the first receptor on a surface of a living cell of the first type in a particular temporal pattern to induce a target response by the living cell. Other techniques for controlling an atomic force microscope comprising a nanoscale tip include controlling the cantilever to cause the nanoscale tip to contact a living cardiomyocyte at a predetermined pressure. The cantilever is also controlled to turn off vertical deflection feedback after contacting the cardiomyocyte and collecting deflection data that indicates a time series of nanoscale vertical deflections of the microscale cantilever caused by the living cardiomyocyte.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of Provisional Appln. 61/418,013, filedNov. 30, 2010, and Appln. 13/307,882, filed Nov. 30, 2011, the entirecontents of which are hereby incorporated by reference as if fully setforth herein, under 35 U.S.C. §119(e).

BACKGROUND OF THE INVENTION

Atomic force microscopy (AFM) was first described in 1986 by a group atStanford and IBM, and quickly found application in investigating thematerial properties of surfaces. AFM was soon applied to biologicallyrelevant samples, including proteins and DNA. Applications of scanningbacteria and viruses followed, and enabled topographic information to becollected at the nanoscale. Atomic force microscopy has made inroadsinto mammalian cell biology only in the past few years by analyzing: 1)nanomechanics, such as the mechanical stiffness of cells or theirsensitivity to mechanical forces; 2) binding kinetics in forcespectroscopy assays; 3) the structure of large macromolecular complexes;4) the physical properties of biopolymers, including DNA, lipids, andproteins as they fold and unravel; and 5) receptor mapping in whichmolecules are localized on biological membranes.

SUMMARY OF THE INVENTION

Techniques are provided for inducing or detecting a response in a livingcell by an atomic force microscope cantilever (dimensions on the orderof 0.1 to 1000×10⁻⁶ meters) with a nanoscale tip (dimensions on theorder of 0.1 to 1000×10⁻⁹ meters).

According to a first set of embodiments, a method comprisesfunctionalizing a nanoscale tip of a microscale cantilever with a firstligand for a first receptor associated with a surface of a first type ofcell. The method further comprises, controlling the cantilever to causethe first ligand on the nanoscale tip to contact the first receptor on asurface of a living cell of the first type in a particular temporalpattern to induce a target response by the living cell.

According to another set of embodiments, a method includes mounting aliving cardiomyocyte on a stage of a microscale cantilever with ananoscale tip. A controller for the microscale cantilever is operated tocause the nanoscale tip to contact the cardiomyocyte at a predeterminedpressure. After turning off vertical deflection feedback, deflectiondata that indicates a time series of nanoscale vertical deflections ofthe microscale cantilever caused by the living cardiomyocyte iscollected.

In other sets of embodiments, a system, apparatus or computer readablemedium carrying instructions is configured to perform one or more stepsof at least one of the above methods.

According to another set of embodiments, an apparatus includes an atomicforce microscope comprising a microscale cantilever on which is disposeda nanoscale tip. The apparatus also includes a stage configured to bemoveably positioned relative to the nanoscale tip of the microscalecantilever in microscale steps. The apparatus further includes aconfocal optical microscope. A sample on the stage is disposed in afocal plane of the confocal optical microscope.

According to another set of embodiments, an apparatus includes an atomicforce microscope comprising a microscale cantilever on which is disposeda nanoscale tip. The apparatus also includes a stage configured to bemoveably positioned relative to the nanoscale tip of the microscalecantilever in microscale steps. The apparatus further includes aconfocal optical microscope, wherein the stage is disposed so that asample on the stage is disposed in a focal plane of the confocal opticalmicroscope.

Still other aspects, features, and advantages of the invention arereadily apparent from the following detailed description, simply byillustrating a number of particular embodiments and implementations,including the best mode contemplated for carrying out the invention. Theinvention is also capable of other and different embodiments, and itsseveral details can be modified in various obvious respects, all withoutdeparting from the spirit and scope of the invention. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way of example, and not by wayof limitation, in the figures of the accompanying drawings and in whichlike reference numerals refer to similar elements and in which:

FIG. 1 is a block diagram that illustrates an example setup forobserving an induced response in a living cell, according to anembodiment;

FIG. 2A is a block diagram that illustrates an example operation of theAFM cantilever with functionalized tip on a corralled living cell inview of a confocal microscope, according to an embodiment;

FIG. 2B is a block diagram that illustrates an example operation of theAFM cantilever on a cardiomyocyte, according to an embodiment;

FIG. 3 is a flow diagram that illustrates an example method formanipulating a living cell using an AFM cantilever, according to anembodiment;

FIG. 4A through FIG. 4D are block diagrams that illustrate exampleeffects of shape of functionalized nanoscale tip, according to variousembodiments;

FIG. 5 is a block diagram that illustrates example intermediate stagesin the production of wells to corral living cells on a stage of an AFM,according to an embodiment;

FIG. 6A through FIG. 6D are micrographs that illustrate example wells tocorral living cells on a stage of an AFM, according to variousembodiments;

FIG. 7 is a block diagram that illustrates an example method tomanipulate immune response of a T-cell of a mammalian immune system,according to an embodiment;

FIG. 8 is a graph that illustrates an example energy diagram that can besolved from AFM dynamic force spectroscopy, according to an embodiment;

FIG. 9A and FIG. 9B are block diagram that illustrate example relationof nanoscale tips of AFM cantilevers to cells corralled in wells in afocal plane of a confocal microscope, according to various embodiments;

FIG. 10 is a graph that illustrates example results from manipulating aliving cell with a functionalized tip of an AFM cantilever, according toan embodiment;

FIG. 11A is a graph that illustrates example force curves of p-MHC on5C.C7 T cell showing extension and retraction of cantilever, accordingto an embodiment;

FIG. 11BA is a graph that illustrates example force spectroscopy ofanti-TCR antibodies on naïve CD4 T cells, according to an embodiment;

FIG. 11C is a graph that illustrates example motile response of amanipulated T-cell, according to an embodiment;

FIG. 12 is a chart that illustrates example ligands for one or morefunctionalized tips useful in manipulating CD4-Tcells, according to anembodiment;

FIG. 13 A is a block diagram that illustrates example signaling byinduced mechanotransduction, according to an embodiment;

FIG. 13B is a block diagram that illustrates an example experimentalsetup to determine effects of mechanotransduction, according to anembodiment;

FIG. 14A and FIG. 14B are flow charts that illustrate an example methodfor an AFM to interact with a living cardiomyocyte, according to anembodiment;

FIG. 15 is a graph that illustrates an example measured force timeseries when the AFM cantilever interacts with the living cardiomyocyte,according to an embodiment;

FIG. 16A through FIG. 16C are graphs which show differences inhistograms of beat force for corresponding cardiomyocytes between thosederived from iPSC and hESC stem cells, according to various embodiments;

FIG. 17A through FIG. 17C are graphs which show differences inhistograms of beat width (duration) for corresponding cardiomyocytesbetween those derived from iPSC and hESC stem cells, according tovarious embodiments;

FIG. 18A through FIG. 18C are graphs which show differences inhistograms of beat rate (frequency) for corresponding cardiomyocytesbetween those derived from iPSC and hESC stem cells, according tovarious embodiments;

FIG. 19A is a micrograph that illustrates an example cluster ofcardiomyocytes, according to an embodiment;

FIG. 19B is a graph that illustrates an example measured force timeseries when the AFM cantilever interacts with a cluster of livingcardiomyocytes, according to an embodiment;

FIG. 19C through FIG. 19E are graphs that illustrate histograms of beatforce, beat width and beat rate, respectively, for a cluster of livingcardiomyocytes, according to an embodiment;

FIG. 20A through FIG. 20D are graphs that illustrate an example effectof norepinephrine on beat force of cardiomyocytes derived from iPSC andhESC-CM stem cells, according to various embodiments;

FIG. 21A through FIG. 21D are graphs that illustrate an example effectof norepinephrine on beat rate of cardiomyocytes derived from iPSC andhESC-CM stem cells, according to various embodiments;

FIG. 22A through FIG. 22C are graphs that illustrate example differencesamong beat force, frequency and duration, respectively, betweencardiomyocytes derived from control patients and diseased patients,according to various embodiments;

FIG. 23 is a diagram that illustrates a micrograph of an example AFMcantilever disposed over an example cardiomyocyte derived from iPSC andan example grid that illustrates a range of a dwell map, according to anembodiment;

FIG. 24 is diagram that illustrates example graphs of beat force andlocal stiffness measurements obtained from dwell maps of cardiomyocytesderived from either a healthy control subject or from a subject withdilated cardiomyopathy, according to various embodiments;

FIG. 25 is a block diagram that illustrates a computer system upon whichan embodiment of the invention may be implemented; and

FIG. 26 illustrates a chip set upon which an embodiment of the inventionmay be implemented.

DETAILED DESCRIPTION

A method and apparatus are described for atomic force microscopemanipulation of living cells. In the following description, for thepurposes of explanation, numerous specific details are set forth inorder to provide a thorough understanding of the present invention. Itwill be apparent, however, to one skilled in the art that the presentinvention may be practiced without these specific details. In otherinstances, well-known structures and devices are shown in block diagramform in order to avoid unnecessarily obscuring the present invention.

An atomic force microscope (AFM) cantilever is prepared by covalently ornon-covalently attaching ligands, and then brought into contact withcells. The cantilever can also be prepared without ligands. The contactis precisely controlled with regards to force applied and duration.Intermittent contacts can be given with high frequency (up to MHz) orlow frequency (minutes, hours of contact time). In some embodiments,each contact involves one or more receptors and corresponding ligands orother components of a functionalized tip. These contacts and receptorligation events cause changes in the recipient cell. For example, it isknown that stem cells differentiate under mechanical forces—thisdifferentiation can be skewed in various embodiments.

In an example embodiment, T cells are used. It is known that T cells canbe activated by receptor ligation. The duration of ligation iscontrolled to skew T cells towards regulatory or activated phenotypes.By ligating other T cell receptors, the differentiation of the T cells(towards different cytokine-secreting phenotypes: Th1, Th2, Th17) can becontrolled. One embodiment is described in the context of inducingcalcium flux as a proxy for activation in T cells. However, theinvention is not limited to this context. In other embodiments the sameor different response of the same or different types of living cells areinduced by contacting a receptor on the cell with the same or differentligands on a nanoscale tip of an atomic force microscope.

For example, stem cells are acted upon in vitro to achieve specificdifferentiation patterns; T cells are acted upon in vitro to becomeregulatory (for transplantation or allergies or autoimmunity); T cellsare acted upon in vitro to alter their differentiation (to fightinfections or tumors); mast cells are acted upon to raise theirthreshold of activation (to reduce allergies); pathogens are acted uponto change the trajectory of infection, or to provide vaccine-typeimmunity; or B cells are acted upon to produce fewer antibodies (forautoimmunity) or antibodies of the “wrong” isotype, and then introducedinto patients for therapeutic effects, among other applications.

In some illustrated embodiments, stem cells are differentiated intocardiomyoctes whose beat characteristics are determined by interactionswith a nanoscale tip of an AFM cantilever, even withoutfunctionalization of the nanoscale tip.

In various embodiments, therapies include administering atherapeutically effective dose of cells modified by or identified by theillustrated methods, or products thereof. Any method of administrationmay be used in various embodiments, including introduction through anyorifice, into any body lumen, or subcutaneously, or intravenously, orsome combination.

Various references are cited herein, each of which is herebyincorporated by reference as if fully set forth herein, except so far asthe terminology is inconsistent with the terminology used herein.

As used herein, the following terms have the meaning provided.

anergy A lack of reaction by the body's defense mechanisms to foreignsubstances. atomic force microscopy A very high-resolution type ofscanning probe microscopy, with demonstrated resolution on the order offractions of a nanometer, more than 1000 times better than the opticaldiffraction limit. Information is gathered by “feeling” the surface witha mechanical probe. Piezoelectric elements that facilitate tiny butaccurate and precise movements on (electronic) command enable the veryprecise scanning. Atomic force A device configured to perform atomicforce microscopy. microscope (AFM) Cardiomyocyte (CM) A cardiac musclecell that beats without external stimulation. chronotrope An agent thatalters the rate of cardiac muscle contractions (positive increase rate,negative decreases rate). confocal microscopy An optical imagingtechnique used to increase optical resolution and contrast of amicrograph by using point illumination and a spatial pinhole toeliminate out-of-focus light in specimens that are thicker than thefocal plane. force spectroscopy A set of observed times to escape a bondfor a corresponding set of applied forces. Human embryonic stem A typeof pluripotent stem cell found naturally in a human embryo. cell (hESC)Induced Pluripotent A type of pluripotent stem cell artificially derivedfrom a non- stem cell (iPSC) pluripotent cell, typically an adultsomatic cell, by inducing a “forced” expression of specific genes.inotrope An agent that alters the force/energy of muscular contractions.ligand A biologically important reagent that interacts with livingcells, such as molecules like p-MHC and antibodies, and including lipidmicelles, viruses, bacteria, small molecules, and even whole cells.microscale A maximum dimension in the range from about 0.1 to about 1000micrometers, μm (also called microns, 1 μm = 10⁻⁶ meters). nanoscale Amaximum dimension in the range from about 0.1 to about 1000 nanometers,nm (1 nm = 10⁻⁹ meters). off rate The rate at which bonds release due tokinetic motions of the bound objects. PDMS Poly-dimethylsiloxanPluripotent stem cell A stem cell that has the potential todifferentiate into any of the (PSC) three germ layers: endoderm(interior stomach lining, gastrointestinal tract, the lungs), mesoderm(muscle, bone, blood, urogenital), or ectoderm (epidermal tissues andnervous system). T cell Thymus-produced cells that belong to a group ofwhite blood cells known as lymphocytes, and play a central role incell-mediated adaptive immunity. Treg (T_(reg)) A specializedsubpopulation of T cells that act to suppress activation of the immunesystem and thereby maintain immune system homeostasis and tolerance toself-antigens.

1. Structural Components

FIG. 1 is a block diagram that illustrates an example setup 100 forinducing and observing an induced response in a living cell, accordingto an embodiment. The system includes an atomic force microscope (AFM)110, and a confocal microscope 120 comprising microscope optics 122 andconfocal light source and detector, such as spinning disk confocalsource and camera 124. In various embodiments, a light source for themicroscope in component 124 is a halogen light, a light emitting diode,or laser, or some combination. A Nipkow spinning disk provides a set ofrotating pinholes for rapidly scanning a viewing area with confocalincident and sample emitted light. Thus, in some embodiments, anapparatus includes a confocal optical microscope.

In other embodiments, e.g., in measuring beat characteristics ofcardiomyocytes described in more detail below, the tip is notfunctionalized with ligands. In such embodiments, a novel apparatusstill includes the AFM, stage for the AFM and confocal microscope. Thus,in some embodiments, an apparatus includes an atomic force microscopecomprising a microscale cantilever on which is disposed a nanoscale tip.The apparatus of such embodiments also includes a stage configured to bemoveably positioned relative to the nanoscale tip of the microscalecantilever in microscale steps. The apparatus also includes a confocaloptical microscope, wherein the stage is disposed so that a sample onthe stage is disposed in a focal plane of the confocal opticalmicroscope.

To successfully assemble this instrument, the vibrational energy fromcontinuous rotation of a Nipkow spinning disk in source and camera 124,which would ruin the sensitive measurements of the AFM, presented achallenge that had to be overcome. This problem was solved using bothpassive vibration control (granite slab 134) and active noisecancellation system 136 (e.g., sensing vibration and producingcountervailing nullifying vibrations at nodes of the granite block).According to some vendors, this combination is aone-of-a-kind-in-the-world tool, and provides the capability to dosimultaneous, high-resolution, fluorescent imaging of live cells inconjunction with molecular manipulation via the AFM. Thus, in someembodiments, an apparatus includes a confocal microscope wherein theconfocal optical microscope is a spinning disk confocal opticalmicroscope and the apparatus further comprises an acoustical isolationcomponent between a stage for the AFM and a spinning disk of thespinning disk confocal optical microscope. In some of these embodiments,the acoustical isolation component further comprises at least one of amassive slab or an active vibration cancellation system

The illustrated embodiment includes a custom metal positioning jacket132 that attaches the source and camera 124 to the granite slab 134 andleaves a spatial gap 124 to avoid transferring vibrations to themicroscope optics 122 and thence to the AFM 110. The spacing wasmanipulated to prevent the spinning disk from touching the microscope.AFM-microscope coupling is per manufacturer.

A computer system 140, such as a system comprising one or more networkedcomputers as described below with reference to FIG. 25, or one or morechip sets in or attached to each component, as described below withreference to FIG. 26, is used to control and collect data from one orboth of the confocal microscope 120 and the AFM 110 or to analyze datatherefrom, or some combination. In the illustrated embodiment, thecomputer system 140 includes a confocal microscope controller andacquisition module 152 to control the confocal microscope 120 andcollect data from it, and an AFM controller and acquisition module 154to control the AFM 110 and collect data from it, and an analyzer 156 toprocess data from one or both of modules 152 and 154. Although computersystem 140 is depicted as having a wired connection to the microscope120 and AFM 110, in other embodiments one or more of these connectionsare wireless. Thus, in some embodiments, a computer readable mediumcarries instructions that cause an apparatus to control the cantileverto cause the first ligand on the nanoscale tip to contact the firstreceptor on a surface of a living cell of the first type in a particulartemporal pattern to induce a desired response by the living cell.

FIG. 2A is a block diagram that illustrates operation of the AFMcantilever 212 on a corralled living cell 290 in view of a confocalmicroscope, according to an embodiment. In the illustrated embodiment, atransparent glass slide 226 rests on microscope stage 222 in view of amicroscope objective lens 224 of microscope optics 122. The glass slidesupports an at-least-translucent, sample stage for the AFM, such astransparent poly-dimethylsiloxane (PDMS) stage 262. In some embodiments,the glass slide 226 serves as the AFM stage; and, in some embodiments,the glass slide 226 is omitted so that the at-least-translucent stagerests directly on the microscope stage 222 In other embodiments, othersuitably transparent or translucent materials are used as AFM stage orstage support. In experimental embodiments, the AFM sits on a stage,held tightly in position because its three legs are placed into threeholes in the stage. That AFM stage allows the specimen (glass slidebearing cells) to be positioned just underneath the cantilever. It doesso by having a region in the center of the AFM stage for the glass slideto sit and be held in place with little permanent magnets placed on topof the slide that are attracted to permanent magnets embedded in thestage. The AFM stage itself can be moved in two horizontal (X, Y)directions by screws and the specimen itself can also be moved, relativeto the stage, using XY piezo motors built into the AFM stage. In theillustrated embodiment, the stage 262 includes one or more microscopicscale wells (called microwells hereinafter) to corral living cells thatmight otherwise move autonomously. In other embodiments the microwells264 are omitted. In the illustrated embodiment, the AFM cantilever 212includes a nanoscale tip 214 functionalized by one or more ligandmolecules 216. Thus, in some embodiments, an apparatus comprises anatomic force microscope comprising a microscale cantilever on which isdisposed a nanoscale tip functionalized with a first ligand for a firstreceptor associated with a surface of a first type of cell. Theapparatus also includes a stage comprising a microscale well forcorralling a living cell of the first type. The stage can be positionedrelative to the functionalized tip of the microscale cantilever. Inembodiments with a confocal microscope, the stage of the AFM is disposedin a focal plane of the confocal optical microscope.

The AFM cantilever with the functionalized tip, and the device toobserve the induced effect in some embodiments that include such adevice, like the confocal microscope, are controlled by a computingsystem 140 using any technology known in the art.

FIG. 2B is a block diagram that illustrates an example operation of theAFM cantilever 272 on a cardiomyocyte 292, according to an embodiment.In this embodiment, the beat characteristics of a living cardiomyocytesare determined, and the effects of various formation and treatment plansare compared to select optimal treatment. In this embodiment, the AFMcantilever 272 is manipulated so that the tip 274 indents thecardiomyocyte 292 attached to an AFM stage 280. The tip 274 need not befunctionalized with any ligands. Then the vertical fluctuations aretracked to determine beat characteristics. By deriving cardiomyocytesfrom different types of stem cells and subjecting the cardiomyocytes todifferent treatments, the viability of different sources ofcardiomyocytes and treatments for cardiac patients can be assessed, asdescribed in more detail in a later section.

In some embodiments, the favored treatments or stemcell-derived-cardiomyocytes, or some combination, are administered to apatient in a therapeutic dose to treat some cardiac ailment, such ascardiomyopathy.

2. Changing a Living Cell

In a first set of embodiments, techniques include functionalizing ananoscale tip of a microscale cantilever with a first ligand for a firstreceptor associated with a surface of a first type of cell. Thecantilever is controlled to cause the first ligand on the nanoscale tipto contact the first receptor on a surface of a living cell of the firsttype in a particular temporal pattern selected to induce a targetresponse by the living cell.

2.1 Method

FIG. 3 is a flow diagram 300 that illustrates an example method formanipulating a living cell using an AFM cantilever, according to anembodiment. Although steps are shown in FIG. 3, and later flow diagramsFIG. 14A and FIG. 14B, as integral blocks in a particular order forpurposes of illustration, in other embodiments, one or more steps orportions thereof are performed in a different order, or overlapping intime as performed in series or parallel, or are omitted, or one or moreadditional steps are added.

In step 301, a nanoscale tip on a microscale cantilever isfunctionalized by attaching, to the tip, a ligand that binds to a targetreceptor on a target type of cell. For example, ligand molecules 216 areattached to tip 214 on AFM cantilever 212. Any method may be used tomake the attachment. Coupling biomolecules to the tip of an AFMcantilever presents two major challenges. First, coupling of themolecules is preferably either covalent or has a binding affinity vastlygreater than the affinities being tested in a binding experiment.Second, the traditional pyramidal geometry of an AFM tip makes thenumber of molecules presented variable based on the degree of cellularcontact.

In an illustrated embodiment streptavidin is coupled to Au-coatedcantilevers using direct chemisorption (see, for example, Ebner A,Wildling L, et al. “Functionalization of probe tips and supports forsingle-molecule recognition force Microscopy,” Stm and Afm Studies On.Berlin: Springer-Verlag Berlin; 2008. p. 29-76). Au-coated cantilevers(from MIKROMASCH™ of San Jose, Calif. or Olympus cantilevers from ATOMICFORCE™ of Mannheim, Germany, spring constant <0.1 N/m) were treated forone minute in oxygen plasma to remove organics and to activate surfacebinding. The cantilever was mounted on a small block ofpoly-dimethylsiloxane (PDMS) for stability. Streptavidin (10 μg/mL) waspipetted directly onto the cantilever and allowed to equilibrate at 4°C. overnight. After rinsing in phosphate buffered saline (PBS),biotinylated antibody (10 μg/mL) was pipetted atop the cantilever andallowed to equilibrate at room temperature for 10 minutes, then washedwith PBS extensively before use. Verification that the conjugationworked was provided by testing the functional capability of theantibodies on the cantilever tip to ligate cell-surface receptors and todo force spectroscopy in embodiments described below.Biotin-streptavidin interactions have been used in many instances forcoupling molecules onto cantilevers. Thus any of multiple ligands can bebound to the streptavidin attached to the nanoscale tip. Therefore, someembodiments include functionalizing a nanoscale tip of a microscalecantilever with a first ligand for a first receptor associated with asurface of a first type of cell.

One concern has been that pyramidal tips present a variable number ofmolecules/contacts to a cell depending on the force applied. Thisproblem makes measurements of affinities and kinetics difficult, becauseof the confounding effect of multivalent interactions between the tipand the cell-surface receptors. In some embodiments, a functionalizednanoscale tip (also called nanoprobe, herein) is used with a flatcantilever tip that presents a fixed number of contacts regardless offorce. FIG. 4A through FIG. 4D are block diagrams that illustrateexample effects of shape of functionalized nanoscale tip, according tovarious embodiments. FIG. 4A depicts a microscale cantilever 412 with ananoscale pyramidal tip 414 functionalized with biomolecules 416. Asdownward force is applied to cantilever 412 a variable number of thebiomolecules 416 are presented to receptors 492 on cell 490. FIG. 4Bdepicts a microscale cantilever 412 with a nanoscale flat tip 424functionalized with biomolecules 416. As downward force is applied tocantilever 412 all of the biomolecules 416 on tip 424 are presented toreceptors 492 on cell 490, the number of which remain fixed no matterwhat additional force is applied. Thus, some embodiments use speciallydesigned nanoprobes for biological applications, such as this nanopillartip designed by the Melosh Group at Stanford University and subsequentlymodified. FIG. 4C is a micrograph that depicts an upper tip 420 with ananoscale pillar disposed below. FIG. 4D is a micrograph that depicts aclose up view of the nanoscale pillar 422 with flat contact surfacebelow. To product this configuration, a microscale tip is mounted withina focused ion beam instrument. The tip is scanned by scanning electronmicroscopy (SEM), which is part of the focused ion beam machine. Usingthe SEM image, gallium ions are directed to sculpt the tip. The galliumbeam simply carves away the silicon of the cantilever. The technique hasbeen published extensively.

Returning to FIG. 3, in step 303, a target cell is corralled in amicroscale well on a stage of the AFM. In some embodiments in which thetarget cell type is not motile, step 303 is replaced with a step tomount a cell of the target type on a different stage, as described belowwith reference to FIG. 14.

Many target cell types are motile, and can crawl away from an AFMcantilever during an experiment. This motility was evident during pilotstudies with AFM and T cells of the mammalian immune system, wherein theT cells crawled away from under the cantilever tip. Immune cells such asT cells use this motility (about 10 microns per minute) to access sitesof infection or inflammation, or while trafficking within the lymphnodes to seek antigenic stimulation. Trying to affix cells to a surfaceby applying integrin ligands such as fibronectin or laminin, or chargedpolymers such as poly-lysine, can partially activate the cells, whichmay interfere with the purpose of an experiment. To prevent motile cellsfrom crawling away during AFM interrogation, a method was devised tocorral the cells by fabricating shallow microwells using PDMS.

In an experimental embodiment, 2 μm deep, 5 μm×5 μm wells werefabricated using soft lithographic techniques. FIG. 5 is a block diagramthat illustrates example intermediate stages in the production of wellsto corral living cells on a stage of an AFM, according to an embodiment.A SU-8 photo resist was spin coated to about 2 to 3 microns thick layer520 on a silicon wafer 510. A chrome mask 530 was positioned above thephotoresist layer 520 and the assembly was exposed to ultraviolet (UV)light 503 to polymerize the exposed photoresist. The chrome mask 530 wasremoved and the un-polymerized photoresist was washed away, leaving apatterned polymerized photoresist layer 522, 2-3 microns thick, to serveas a mold on the silicon wafer 510. Liquid PDMS was poured over the moldto form a PDMS layer 540 with a thickness of greater than 2 microns.When set, the PDMS layer 540 is peeled off the mold and attached, wellside up to a transparent substrate support 550, such as glass.

FIG. 6A through FIG. 6D are micrographs that illustrate example wells tocorral living cells on a stage of an AFM, according to variousembodiments. FIG. 6A is a scanning electron microscope (SEM) image 610with horizontal and vertical scales given by tick marks 612 indicating50 microns. The micrograph 610 depicts a PDMS stage 640 for an AFM withan array of microwells 642. FIG. 6B is a scanning electron microscope(SEM) image 620 with horizontal and vertical scales given by tick marks622 indicating 4 microns. The micrograph 620 depicts a single microwell642. FIG. 6C is a confocal microscope image 630 that depicts an array ofmicrowells 642 with individual T cells 650 corralled in severalmicrowells. FIG. 6D is a total internal reflection (TIRF) microscopybright field image 670 taken through a confocal microscope. Thehorizontal axis 672 and vertical axis 674 indicate distance in microns.Micrograph 670 depicts the array of microwells 642 as well as T cells650, including a T cell (image blocked) while the T cell is beinginterrogated by a tip of an AFM cantilever 660.

During step 303, cells of the target type are pippetted onto the stagewith microwells. For example, in an experimental embodiment, primarymouse CD4 T cells were disposed by pipette over the top of the wells andwere allowed to settle at 37° C. for 30 minutes. These were imaged, suchas depicted in FIG. 6C. Experiments described in more detail belowconfirmed that cells were still functional when corralled in suchmicrowells. For example, a T cell that was restrained from crawling awayby a microwell was able to be interrogated by AFM for 20 to 30 minutes.Thus, in some embodiments, a method further comprises corralling theliving cell in a microscale well.

It is also a challenge to keep cells alive on the AFM stage for suchmanipulation. A number of difficult challenges arise when using AFM withliving cells. First, the cells require a warm and nutrient-richenvironment. Use of a submersible heating element keeps the cells warm,but can create thermal currents that affect the deflection of the AFMcantilever. In various embodiments, this challenge is addressed by 1)allowing the cantilever to equilibrate in warm media for about 30 toabout 60 minutes before work, and 2) by developing AFM cantilevers thatare metal coated on both sides, so as to decrease the effects ofasymmetric thermal expansion.

Returning to FIG. 3, in step 305 the AFM stage is mounted below themicroscale cantilever so that the stage and cantilever can move relativeto each other in nanoscale increments. Either the cantilever or thestage, or both, move relative to a laboratory frame of reference. Suchincremental movement is routine for many currently available AFMs knownin the art. The nanoscale increments allow the nanoscale tip to bepositioned to contact particular receptors on a cell membrane of a cellaffixed to the stage, such as a living cell disposed within a microwell.

The scan rates of widely available AFMs are from about 0.1 Hz to about 1Hz. Scan rate in Hz represents the time to scan one line of the desiredarea. The desired area may be divided up into some number of pixels in Xand Y. The AFM piezo stage will then scan the desired area. Scan ratetells how fast it can scan once across X. Such scan rates consumeminutes to scan a cell-sized area, during which time a cell can changesignificantly. Thus, cells should be kept alive and functioning for suchtime scales after mounting. This last issue is less of a concern forsome embodiments in which faster scanning is not involved.

In some embodiments, the AFM stage is mounted in the focal plane of aconfocal microscope system, such as confocal microscope 120. In suchembodiments, the method 300 of FIG. 3 includes steps 307 or step 308, orboth. For example, in embodiments that determine the response of a cellto manipulation by the AFM, fluorescent molecular probes are often used,which probes are detected by the confocal microscope system or someother microscope system.

In step 307, the AFM stage and cantilever with functionalized tip aremounted above the objective lens of a confocal microscope, such aconfocal microscope 120. If the confocal microscope has a source ofvibration, e.g., spinning Nipkow disc, which would affect the motion andoperation of the AFM, then, in step 309, the cell stage is isolated fromthese vibrations. For example, the spinning disk portion 124 is placedseparately on a granite slab 134 with a spatial gap 124; and, any activevibrational controls 136 are turned on.

In step 311, the nanoscale tip is controlled so that a ligand of thefunctionalized tip contacts a receptor on the cell in a particulartemporal/spatial pattern. For example, a ligand on the functionalizedtip interacts with one or more receptors on living cells in asingle-ligand-on-single-receptor fashion with precise on-off control.This use of AFM to ligate receptors on live cells with spatiotemporalcontrol is an innovation that has the potential to expand knowledge ofthe function of receptors on immune cells and eventually provide thecapability to “program” the differentiation pathways of T cells, stemcells, or other cells ex vivo, for cellular therapy or for fundamentalstudies. This step delivers ligands to cell-surface receptors, toactivate these receptors in a temporally- and spatially-precise way, andto either affect or determine the downstream biology of single receptorligation, or both. With respect to target T-cell types, this novelapproach is expected to give new quantitative and qualitativeunderstanding of T-cell activation, and also yield new therapeuticstrategies to shape T-cell responses in cancer, autoimmunity,transplantation, allergy, and infection. Thus, in some embodiments, amethod includes controlling the cantilever to cause the first ligand onthe nanoscale tip to contact the first receptor on a surface of a livingcell of the first type in a particular temporal pattern to induce adesired response by the living cell.

FIG. 7 is a block diagram that illustrates an example method tomanipulate immune response of a T-cell of a mammalian immune system,according to an embodiment. AFM cantilever 712 is coupled through afunctionalized tip with antibodies 716 that ligate receptors 791 on a Tcell 790 restrained in a microwell 264 in a stage, such as PDMS stage262. By controlling the height through z-axis piezo movement 714 withtime, the T cell receptors 791 are gently ligated and released in atemporal pattern of choice. The cantilever can then be movedhorizontally to ligate gently a different receptor of the same ordifferent type on the same cell or a different cell in a differentmicrowell.

In some embodiments, the pattern of gentle ligation is based on forcespectroscopy, which is obtained from the literature or from other AFMexperiments or, in some embodiments, experiments using the sameequipment. In the equilibrium between free receptor and ligand and theirbound state, according to the Arrhenius model, an energy barrier must beovercome for the reaction to proceed. According to the Bell model (BellG., Science; v 200 (4342) p618, 1978) as modified by Evans and Ritchie(Evans E, Ritchie K., Biophys J.; v72(4) p1541-55, 1997. PMCID:1184350), applying an external pulling force f to a receptor-ligandinteraction exponentially lowers the transition state energy barrier andincreases the dissociation rate k_(off)(f) as given by Equation 1.

where k⁰ _(off) is the dissociation rate in the absence of a pullingforce, γ is a the position of the transition state energy barrier (inAngstroms of bond separation), T is absolute temperature, and k_(B) isBoltzmann's constant.

The AFM is capable of applying a constant loading force to the bond byretracting the cantilever tip at a rate r, giving the most probableunbinding force f_(max) for the unbinding of the complex to be given byEquation 2 (Wojcikiewicz E, Abdulreda M, et al. Biomacromolecules,v7(11), pp 3188-95, 2006).

$\begin{matrix}{{f_{\max}(r)} = {\frac{k_{B}T}{\gamma}{\ln \left( \frac{\gamma \; r}{k_{off}^{0}k_{B}T} \right)}}} & (2)\end{matrix}$

By measuring the unbinding force at a variety of pulling rates, theoff-rate (k_(off)) can be derived at a range of forces, and thus theoff-rate calculated in the absence of force. Nonlinear fitting of theobserved forces plotted against a variety of pulling rates yields theposition of the energy barrier γ distance in Angstrom. The reduction inthe height of the transition state energy barrier ΔΔG(f) is thencalculated from Equation 3, and depicted in FIG. 8.

$\begin{matrix}{{{\Delta\Delta}\; {G(f)}} = {{{\Delta \; G^{0}} - {\Delta \; {G(f)}}} = {{- k_{B}}T\; {\ln \left( \frac{k_{off}^{0}}{k_{off}(f)} \right)}}}} & (3)\end{matrix}$

FIG. 8 is a graph 800 that illustrates an example energy diagram thatcan be solved from AFM dynamic force spectroscopy, according to anembodiment. The horizontal axis 802 is reaction coordinate (related todistance between ligand and receptor) in Angstroms (Å, 1 Å=10⁻¹⁰meters). The vertical axis 804 is energy of the reacting molecules(related to absolute Temperature and rate of applying force with the AFMcantilever) in relative units. Trace 810 indicates the energy state ofthe two molecules at various distances with a particular force appliedby the AFM as related to the off rate k_(off). The trace 820 indicatesthe energy state of the two molecules at various distances at the limitof no force applied by the AFM which is related to k⁰ _(off). The energyminimum associated with the binding state of the two molecules isindicated by bound complex position 840, and the energy state of the twomolecules separated at distance is indicated by the dissociated complex842. The energy barrier that must be overcome to separate the moleculesis at position γ 844 and the height of the barrier is given by AG(f) 830when a force is applied by the AFM and ΔG⁰ 832 without such a force. Thedecrease in energy threshold when the force f is applied is given byΔΔG(f) 834, as computed using Equation 3, above.

Thus dynamic force spectroscopy can be used to calculate all aspects ofreceptor-ligand kinetics, but with the unique advantage of measuringkinetics on the live cell, rather than in an artificial environment.With knowledge gained by experiment of the force to apply to thecantilever to bind and dissociate any ligand to any receptor, thenumber, duration and rate of binding of the ligands on thefunctionalized tip to receptors in a cell membrane can be controlled andused to obtain or determine cell response to such stimulation.

In some embodiments, the response of the cell is determined in step 313.In some embodiments, the response is determined by performing chemicalanalysis or sequencing operations on the material in cells manipulatedby the functionalized tip. Thus, in some embodiments, a method includesdetermining an effected response by the living cell.

In some embodiments, the response is determined during step 313 byoperating the confocal microscope and any vibrational cancellationsystem in step 315. For example, in some embodiments, the response isobserved using a microscope, such as confocal microscope 120 in system100, in step 315. The apparatus of FIG. 1 is especially useful indetermining this response, because the cell being manipulated by the AFMis directly observable by the confocal microscope. FIG. 9A and FIG. 9Bare block diagram that illustrate example relation of nanoscale tips ofAFM cantilevers to cells corralled in wells in a focal plane of aconfocal microscope, according to various embodiment. FIG. 9A is acombined total internal reflection (TIRF) microscopy and bright fieldimage 910 taken through a confocal microscope with horizontal axis 902and vertical axis 904 indicating relative distance in microns. Themicrograph 910 depicts microwells 942 on an AFM stage and a microscalecantilever 960 on which is drawn an outline of the cantilever tip 962. Acell 950 corralled in a microwell is being manipulated by this tip 962.FIG. 9B is a combined bright field, confocal fluorescence, and TIRFmicroscopy image 970 of an AFM cantilever 980 and functionalized tip 982next to a primary mouse T cell 990. The distance scale is indicated byhorizontal bar 972. The confocal image shows fluorescence emission fromthe T cell 990 in a red band by calcein red-orange, which is acytoplasmic dye, and a blue band by Hoechst, which is a nuclear DNA dye.As manipulation by the functionalized tip 982 affects the T cell 990,the fluorescent emission can be varied and observed by the confocalmicroscope even absent the bright field and TIRF imagery. Thus, in someembodiments, determining an effected response by the living cell furthercomprises operating a microscope to image the living cell. In some ofthese embodiments, operating the microscope to image the living cellfurther comprising reducing vibrations from the microscope from reachingthe living cell or microscale cantilever.

In some embodiments, the response is already known from previousexperiments to be a target response (e.g., acquired immune response ofthe T cell), and step 313 is omitted. In such embodiments, the result ofstep 311 is the programmed cell, e.g., the target cells derived from the“programmed” stem cell or the T-cell “programmed” for a target immuneresponse, or an antibody produced from such a programmed T cell.

Returning to FIG. 3, in step 317, one or more cells with the properresponse, e.g., a differentiated cell from a stem cell or an immuneresponse to a particular antigen, or a product of such a cell, isadministered to a subject in a therapeutic dose that interrupts orretards the progress of a disease.

The lack of in vitro tools that enable instantaneous on-and-off controlof cellular receptor signaling has been a serious hindrance indeveloping a quantitative understanding of how cells process signals.Understanding how T cells process their receptor signals in their“decision” to become activated or tolerant may be an important questionfor many cell types: neurons (how transient action potentials lead overtime to long term potentiation and memory), pancreatic beta cells (howbrief ion channel signals accumulate to the decision to releaseinsulin), and endothelial cells (how days of mechanical wall pressure inblood vessels alters the physiology of high blood pressure). These kindsof questions provided the impetus to develop the described novelbioengineering approach using AFM to study signal integration.Techniques in nanotechnology, including soft lithography and AFM, wereinvolved to develop this approach, but just as important were the deepexperiences in immunology, cell biology, and biophysics that allowedidentification of the important, long-standing questions.

Much as the invention of optical tweezers enabled the control ofmolecule-laden beads or cells, and led to an explosion insingle-molecule biophysics, and the invention of AFM enabled anexplosion in the materials sciences, the development of the technologypresented here shares three key features: i) availability; ii) multiplyapplicable; and iii) customizable. With regard to availability, almosthalf of the AFM units sold by ASYLUM RESEARCH™ of Goleta, California goto biology labs, so many labs are already poised to apply theseapproaches to the cell types they study. With respect to multipleapplications, it is shown that, not only, are these techniquesapplicable to three long-standing puzzles in T cell biology describednext, but also these techniques apply to many kinds of cells. Withregard to customizable, not only is it shown that biological moleculessuch as p-MHC and antibodies can be coupled to the AFM cantilever, butalso attachment of other biologically important reagents to the AFM canbe envisioned, including lipid micelles, viruses, bacteria, smallmolecules, and even whole cells. This customizability allowsinvestigators to use biological AFM to address long-standing problemsacross cell biology.

2.2 Example Embodiments

Partial observations from various example embodiments were describedabove to illustrate the steps of method 300. Here various embodimentsare described in more detail.

T cells require ligation of the antigen-specific T cell receptor (TCR)by an antigenic peptide associated with the major histocompatibilitycomplex (abbreviated p-MHC hereinafter) to become activated. When thecell surface costimulatory receptor CD28 is also ligated at the sametime as TCR, the amount of antigen needed to activate the T cell isgreatly reduced and the immune response is learned. CD28 dramaticallyfacilitates the survival and cytokine production of activated T cells,and helps shape the trajectory of differentiation to the various helperT cell subsets (Th1, Th2, Th9, Th17, etc.). The inhibitory receptor PD-1plays an opposite role: when co-ligated along with TCR, PD-1 decreasescytokine production and arrests the cell cycle. In the presence ofexogenous TGF-β, ligation of PD-1 on T cells drives the differentiationof helper T cells into regulatory T cells. T cell activation andeffector behavior may be controlled by many such cell-surface receptors,including LFA-1, PD-L1, CTLA-4, NKG2D, and ICOS. How the T cellintegrates dozens of contemporaneous “positive” and “negative” signalsover the many hours of its myriad encounters with APCs in the lymph nodeis as yet unknown, and can be discovered using the techniques describedhere.

Ligating both TCR and costimulatory receptors on T cells with precisecontrol by AFM enables activating antigen-specific T cells in vitrousing a specific pattern of signals that enables reprogramming of thedifferentiation pathway, and then re-transferring back to the host. Ithas been shown that adoptive transfer of even a single T cell canmediate delayed type hypersensitivity in the skin and protection frominfection. It is anticipated that combining AFM reprogramming and invitro expansion creates a large number of specifically programmed cells,constituting a novel regimen of cellular therapy that could play animportant role in tumor immunotherapy or in severe autoimmune disease.Furthermore, this technique can be applied to differentiate stem cells,which require both receptor signals as well as mechanical forces toshape their lineage decisions.

The affinity of the TCR for its cognate p-MHC is quite weak (lowmicromolar dissociation constant), mainly due to a fast off rate. Thedevelopment of “altered peptide ligands”, that is, peptides with aminoacid substitutions of the wild-type sequence that still bind the TCR butwith altered affinity (either higher or lower than wild-type), hashighlighted the importance of binding kinetics of the cognate peptidefor T cell activation. There have been conflicting studies through theyears identifying the off-rate or the affinity as the key kinetic factorthat relates to T-cell outcomes. Altered peptide ligands have been usedto accelerate T-cell responses in cancer therapy, or, on the flip side,to skew T-cell differentiation pathways as a treatment for autoimmunity,or to induce T-cell anergy (unresponsiveness) or regulatory T cells.What is lacking currently is the ability to predict how a mutant peptidewill affect the outcome of T-cell activation—addressing this problem isperformed in some embodiments.

In experimental embodiments described here, T cells are manipulatedusing the AFM. Antibodies specific for the mouse T cell receptor werecoupled onto a nanoscale tip of a microscale cantilever usingstreptavidin-biotin linkage, as described above. Thus, in someembodiments of the methods, the first type of cell is a T-cell of amammalian immune system, and the desired response by the living cell isan immune response to the ligand. As described above, in some of theseembodiments, the method further comprises administering the living cellto a subject to induce in the subject an immune response to the ligand.

2.2.1 Stimulated Ca Flux in T-cells

Helper mouse CD4 T cells were loaded with a Ca²⁺-sensitive fluorescentdye (Fluo-4 available from Invitrogen, Carlsbad Calif.), plated onto thesurface of the PDMS microwells, and allowed to settle. Transient, gentlecontact (about 0.05 to 0.5 nanoNewtons, nN, 1 nN=10⁻⁹ Newtons) of thefunctionalized AFM cantilever with the T cell revealed Ca²⁺ influxwithin about 6 seconds of contact as compared to a nearby bystandercell, consistent with many previous studies that Ca²⁺ flux is anessential and early marker of T-cell activation. Lifting the cantileverto unligate its receptor contacts revealed rapid Ca²⁺ homeostasis.

FIG. 10 is a graph that illustrates example results from manipulating aliving cell with a functionalized tip of an AFM cantilever, according toan embodiment. The horizontal axis 1002 indicates time in seconds. Thevertical axis 1004 indicates amount of fluorescence, in arbitrary units,of the Ca²⁺-sensitive fluorescent dye. The time during which the AFMgently ligates the T cell is indicated by bar 1006. Points 1010 form atrace that indicates the Ca²⁺ flow in a T cell that was stimulated bythe functionalized tip of an AFM cantilever. Points 1020 form a tracethat indicates the Ca²⁺ flow in a bystander T cell that was notstimulated by the functionalized tip of an AFM cantilever. Clearly, theCa²⁺ flux increases due to the gentle contact of the functionalized tip.Removal of the tip causes the Ca²⁺ flux to drop below un stimulatedlevels.

2.2.2 Force Spectroscopy on T-Cells

The capability to do force-spectroscopy on T cells was alsodemonstrated. FIG. 11A is a graph that illustrates example force curvesof p-MHC on 5C.C7 T cell showing extension and retraction of cantilever,according to an embodiment. The horizontal axis 1102 indicates vertical(Z) displacement in microns of the cantilever The point of contact ofthe T cell is shown towards the right of the graph where the curveslopes upwards (showing contact and then indentation of the T cell). Thevertical axis 1104 indicates the force required to achieve thatdisplacement in nanoNewtons (nN). The top trace 1120 indicates the forcecurve as the cantilever is extended toward the T cell. The bottom trace1110 indicates the force curve as the cantilever is retracted fromcontact with the T cell. Shaded area from about displacement 3.18microns to about displacement 3.23 microns shows the work ofde-adhesion, which is caused by the breakage of TCR-p-MHC bonds. Theadhesion is measured, and demonstrates force can be used to breaknon-covalent bonds between molecules as predicted by the Bell Equationsgiven above. How much force is needed to rupture the bonds is related toloading rate of pulling on the bond by the force spectroscopy equation,Equation 2, above.

FIG. 11BA is a graph that illustrates example force spectroscopy ofanti-TCR antibodies on naïve CD4 T cells, according to an embodiment.The horizontal axis 1154 indicates force in nanoNewtons and the verticalaxis 1154 indicates counts (e.g., number of events). Bars indicatemaximum off rate at each applied force. Trace 1160 indicates a linearfit to the average off rate. The maximum off rate occurs with a force of0.93 nN (930 picoNewtons, pN, 1 pN=10⁻¹² Newtons). This indicates that0.93 nN of adhesive force is due to the TCR-antibody contact.

2.2.3 Motile Response of T-Cells

FIG. 11C is a graph 110 that illustrates example motile response of amanipulated T-cell, according to an embodiment. The horizontal axis 1172indicates time in seconds. The vertical axis 1174 indicates force innanoNewtons. Trace 1180 indicates the force applied to the cantilever todeflect the functionalized tip sufficiently to bind to the TCR on the Tcell. The contact and indentation of the cell occur at the very leftedge of trace 1180. The trace then shows the responses of the cellmeasured over time as pushing and pulling on the cantilever. Pullingdownward is low on the vertical axis 1174, whereas pushing is up on theaxis 1174, relative to the starting point. The negative force observedat 300 to 400 seconds and again after about 450 seconds (force valleys1182) is taken to indicate that the T cell reaches out to push towardthe functionalized tip, thus indicating a T cell motility response tomanipulation. It is believed that this is consistent with T cellbehavior that fosters increased contact with a potential invader toavoid too brief a contact that might delay an appropriate immuneresponse. Thus, the T cell responds to local ligation (triggering) ofits T cell receptors by mechanically pushing upon the antigen presentingcell to improve the surface area and quality of contact. Thus, the Tcell can become more activated than in a random encounter with aninvader, and more effectively scan particles that are filtered through alymph system. It is anticipated that by being able to deliver localligation plus force in a particular temporal pattern signaling into theT cell can be optimized.

2.2.4 Effects of Altered Ligand on T-Cell Activation

In some embodiments, the effects of different peptides on the integratedsignaling of T cells are determined.

For example, it is known that point mutants of a cognate peptiderecognized by the TCR can active a T cell, but with altered kinetics oroutcomes. For example, these mutations may convert an agonist peptide toa stronger agonist or to an antagonist peptide. Ligation of T cells withantagonist peptides can induce antigen-specific anergy or regulatory Tcells. Exploiting altered peptide ligands has become an importanttherapeutic strategy in cancer immunotherapy and allergy modulation;thus, it is imperative to understand and predict the responses of Tcells to altered peptide ligands. This is performed in some embodiments.

It has long been presumed that the different kinetics of binding ofvariant peptides, as compared with the wild-type peptide, is what drivesaltered T cell responses. However, fifteen years of studies documentingaltered binding kinetics by surface plasmon resonance have been unableto consistently predict the responses of T cells. Even as recently asthis year, new approaches to measure the kinetics of pMHC binding toTCRs are still being sought to improve understanding as to why alteredpeptide ligands change the responses of T cells. However, even recentlypublished approaches have a fundamental flaw because they did notmeasure nanoscale interactions—the number of TCRs ligated on a T cellwas estimated from the surface area of a large bead, and errors in thisestimate could significantly bias the calculated affinities.

FIG. 12 is a chart that illustrates example ligands for one or morefunctionalized tips useful in manipulating CD4-Tcells, according to anembodiment. The chart lists a peptide identifier 1210 for each ofseveral peptides, along with an associated amino acid sequence 1220 anda T cell signaling role 1230 anticipated for the peptide. The top lineindicates an amino acid sequence for wild-type peptide antigen for CD4 Tcells from the 5C.C7 T cell receptor transgenic mouse, and the next fourlines indicate closely related altered peptide ligands in which thesequence 1220 indicates only the deviations from the wild type MCCpeptide sequence. These ligands have variable effects in culture aslisted in the role column, and their effects on signal integration aretested in some embodiments.

Such embodiments solve this problem definitively by using AFM to measurethe binding kinetics of a single TCR to p-MHC using force spectroscopy.In addition to the definitive measurements of kinetics obtained by AFM,this approach has the added advantage of continuously imaging themanipulated T cell directly during activation, so that a physiologicalcorrelate of signaling can be measured immediately and directly.

This embodiment includes covalently attaching p-MHC complexes onto sharpAFM cantilevers with functionalized tips, whose surface area at the tip(about 1-2 nm diameter) allows for a single p-MHC complex to bepresented. Altered peptide ligands that bind the 5C.C7 T cell receptorare listed in FIG. 12. In these embodiments, one or more of the p-MHCsof FIG. 12 are coupled onto the cantilever tip, and CD4 T cells obtainedfrom spleens of 5C.C7 TCR transgenic mice are manipulated. One step isperformed to establish whether lower affinity ligands (102S) activate Tcells as rapidly as the wild-type peptide. Using the z-piezo of the AFM,this AFM cantilever is brought into gentle contact with a 5C.C7 CD4 Tcell with about 10 pN of force, which has been found to be sufficient toligate the TCR but not to induce mechanical strain on the T cell. Thecantilever is retracted at a variety of loading rates and the adhesionforce is measured, as described above with reference to d FIG. 11B.

During contact, the T cell is monitored by spinning-disk confocalmicroscopy, whereby the whole cell can be imaged in about 1 second. Thusreal-time images of Ca²⁺ influx is measured and collected. In someembodiments, movies are collected by analyzing Ca²⁺ flux (Fluo-4fluorescence) normalized for dye loading using calcein red-orange atmultiple times. Next, the antagonistic peptides (102G and 99R) aretested for their capacity to induce T-cell activation. In prior studiesthese peptides induced no Ca²⁺ flux when presented by antigen presentingcells for 3-10 minutes, but were not able to be studied in asingle-molecule fashion. The 93E99T102A peptide is used as a control; itdoes not activate T-cell Ca²⁺ flux, but binds to the TCR.

2.2.5 Costimulatory Signaling of T-Cells

Ligation of the costimulatory receptor CD28 on T cells has been wellestablished to lower the threshold for T-cell activation, as measured bya lower requirement for antigen by CD4 T cells. Similarly, ligation ofthe inhibitory receptors CTLA-4 or PD-1 raise the threshold for T-cellactivation. Ligation or blockade of these pathways has become a veryimportant therapeutic approach in cancer medicine and autoimmunity.

The effects of costimulatory pathways on T cells have been measuredusing traditional tools of cellular immunology (e.g., mixing cells in96-well plates and measuring proliferation or cytokines after asufficient time has elapsed) that, because of ensemble blurring, do notallow understanding of how much of an effect each receptor ligation hason the threshold for activation. Simple questions like how many“stimulatory” units of co-signaling (e.g., CD28 ligation) are needed tocounteract an “inhibitory” unit of co-signaling (e.g., PD-1 ligation)cannot be answered by those traditional approaches, but arefundamentally important to tune T-cell differentiation programs. Aquantitative understanding of how T cells interpret costimulatorysignals is anticipated to improve capability to therapeutically modifythe threshold for T-cell activation.

In this embodiment, p-MHC complex plus either anti-CD28 monoclonalantibodies (mAbs) or anti-PD-1 mAbs are covalently attached onto thetips of AFM cantilevers. CD4 T cells from the 5C.C7 mouse are loadedwith 5 μM Ca²⁺ fluorescent dye Fluo-4 (and co-loaded with a Ca²⁺insensitive dye such as calcein red-orange to normalize dye loading.Naïve T cells are ligated as above. Ca²⁺ flux is measured in threesituations: 1) with p-MHC alone on the cantilever, 2) with p-MHC plusanti-CD28, and 3) with p-MHC plus anti-PD-1. It is already known fromensemble analyses that Ca²⁺ flux is influenced by CD28 ligation and PD-1ligation, so it is assured that Ca²⁺ flux is a valid read-out for thealterations on the threshold for T-cell activation. This approach givesa quantitative readout of the single-molecule effects of costimulation,both stimulatory and inhibitory, and allows development of a model forsignal integration in T cells.

2.2.6 Role of Mechanical Forces on Receptors in Signaling of T-Cells

In this embodiment, AFM manipulation is used to test the hypothesis thatmechanical displacement or torsion of the TCR drives CD3 signaling.

The link between ligation of the TCR by p-MHC and signaling has beenelusive, because all known downstream signals are initiated by the CD3chains associated with TCR. But, it is unknown how TCR binding conveys asignal to CD3. It has been long known that monomeric p-MHC are unable toactivate T cells, but oligomers (including tetramers) are able to.Recent work has supported the notion that mechanical forces, either fromAPC movement or from torsional bending induced by the p-MHC oligomerinduce conformational changes in the TCR-CD3 complex that drivedownstream signaling event, due to tilting or bending a domain of theCD3E chain away from the membrane to expose tyrosines in theimmunoreceptor tyrosine associated activation motifs (ITAMs) forphosphorylation. FIG. 13A is a block diagram that illustrates examplesignaling by induced mechanotransduction, according to an embodiment.FIG. 13A depicts a T cell 1390 with receptors 1350. Monomericinteraction with a ligand is depicted in portion 1310 of the T cellsurface, and is not associated with tilting or bending of the receptor1350. Multimeric interaction with a ligand is depicted in portion 1320of the T cell surface, and is associated with tilting or bending of thereceptor 1350. It is anticipated that interaction with a functionalizedtip of an AFM cantilever, called AFM-coupled interaction herein,depicted in portion 1330 of the T cell surface, will emulate multimericinteraction.

In this embodiment, the AFM cantilever is used to deliver a mechanicalsignal to a single TCR. A p-MHC that can be covalently UV-crosslinked toa TCR (by virtue of an aryl azide group attached by a short flexiblelinker to the end of the peptide) is used to functionalize a tip of theAFM cantilever. FIG. 13B is a block diagram that illustrates an exampleexperimental setup to determine effects of mechanotransduction,according to an embodiment. FIG. 13B depicts the AFM cantilever 1310with tip 1312 functionalized by a functional component 1313 composed ofan aryl azide crosslinker 1318 connected by biotinlated peptide MHC 1316to the streptavidin 1314 biomolecule affixed to the tip 1312. Thisfunctional component 1313 binds to a TCR-CD3 complex 1392 on a T cell1390 restrained in a PDMS microwell 1364 in a PDMS stage 1362 to form alink 1372.

In this embodiment, the tip 1312 functionalized with this p-MHCfunctional component 1313, is controlled to gently touch the T cell. Thecrosslinker is polymerized to the receptor by UV exposure indicated byUV waves 1370 (e.g., for about 30 seconds), at a typical photon doseused for microscopy, to form link 1372. The AFM cantilever is controlledto deliver small lateral/vertical displacement or torsional forces 1382to induce motions to the link 1372 and receptor while continuouslyimaging the T cell for evidence of activation. A broad range ofdisplacements (in a range from about 0.5 nm to about 90 μm) along with abroad range of forces (in a range from about 1 pN to about 10 nN) arepossible with an example AFM. Activation is assessed by measuring Ca²⁺flux 1384 by confocal microscopy, as described above. For example theactivated T cell 1391 fluoresces with Fluo-4 fluorescence in an amountrelated to the calcium flux 1384.

The impact and significance of this technology to study receptor biologyon live cells are far-reaching, since current methodologies to studyreceptor signals limited to traditional ensemble analyses (e.g., 96-wellplates) or cannot target single molecules (e.g., optical trapping ofbeads or cells). In addition, the embodiments described herein providethe first experimental evidence of single-molecule kinetics of the TCR,while at the same time allowing confocal live imaging of the effects ofsingle-TCR activation. Furthermore, the experimental embodiments alsodescribed here provide the basis for cellular reprogramming using AFM.Exciting anticipated embodiments for this technique include: inductionof antigen-specific T cell anergy in transplantation, provision ofpre-differentiated T cells in severe infection responses, or programmingof mechanically sensitive stem cells to particular lineage fates. Theresults of these embodiments provide insight for the future for bothreductionist in vitro experiments, as well as for in vivo studies inregenerative medicine, infectious disease, and translational immunology.

3. Interacting with Living Cardiomyocyte

In another set of embodiments, techniques include mounting a livingcardiomyocyte on a stage of a microscale cantilever with a nanoscaletip. A controller for the microscale cantilever is operated to cause thenanoscale tip to contact the cardiomyocyte at a predetermined pressure.After turning off vertical deflection feedback, deflection data thatindicates a time series of nanoscale vertical deflections of themicroscale cantilever caused by the living cardiomyocyte is collected.In some of these embodiments, the beat characteristics of the livingcardiomyocytes are used to assess development of replacementcardiomyocytes and other treatments of cardiac disease.

Diseases of cardiomyocytes, either primary (e.g., geneticcardiomyopathies) or acquired (e.g., myocardial infarction), are ofmajor importance to health across the world. Understanding thephysiology and pathophysiology of these vital cells has been the subjectof research for over two centuries. Obtaining human biopsy specimensfrom diseased patients, however, requires expensive and invasiveprocedures, which may be poorly tolerated by children or the criticallyill. Recent breakthroughs in induced pluripotent stem cells (iPSC) andin genetic engineering of human embryonic stem cells (hESC) have madehuman disease-specific cardiomyocytes available for elucidatingmechanisms of specific cardiac diseases. To understand themechanobiological properties of these stem cell-derived cardiomyocytes,embodiments were developed to measure contractile forces, beatfrequencies and durations, and membrane stiffness of live, beatingcells.

These techniques use atomic force microscopy (AFM) to quantify themechanobiological properties of pluripotent, stem cell-derivedcardiomyocytes, including contraction force, rate, duration and membranestiffness. Beats were measured from cardiomyocytes derived from inducedpluripotent stem cells and from embryonic cells of healthy subjects andthose with dilated cardiomyopathy. It is found that these AFM techniquescould quantify beat forces of single cells and clusters ofcardiomyocytes, and could detect the inotropic (increase in force ofcontraction) effect of the drug norepinephrine. Cardiomyocytes derivedfrom subjects with dilated cardiomyopathy show decreased force anddecrease membrane stiffness compared to controls. The AFM-basedtechniques described here can serve as a screening tool for thedevelopment of cardiac-active pharmacological agents, as a platform forstudying cardiomyocyte biology, or as a mechanism to fine-tune thechoices of treatments for patients in heart failure.

AFM has been used to study cardiomyocytes in the past, but some of theseefforts required synchronizing the z-piezo of the AFM with beating ofthe cardiomyocytes, which created fluidic disturbances that preventedaccurate measurement of contraction forces. The techniques describedhere touch the cell gently with the AFM cantilever, then lock thez-piezo, which forces contractions of the cell to deflect thecantilever, which deflections are measured. Multiplying the measureddeflection by the spring constant allows calculation of the contractionforce.

3.1 Method

FIG. 14A and FIG. 14B are flow charts that illustrate an example method1400 for an AFM to interact with a living cardiomyocyte, according to anembodiment.

In step 1401 a spring constant for a microscale cantilever isdetermined. Any method known in the art may be used. For example, in anillustrated embodiment, the spring constant of the AFM cantilever wascalibrated using the thermal noise method described in Hutter, J. L.,and Bechhoefer, J., “Calibration of Atomic-Force Microscope Tips,”Review of Scientific Instruments, v64, pp 1868-1873, 1993. The typicalspring constant for these cantilevers was around 0.04 Newtons per meter(N/m) for deflections up to hundreds of nanometers. Thus, in someembodiments, a method includes calibrating a spring constant for themicroscale cantilever.

In step 1403, at least one cardiomyocyte is grown on a stage for theAFM. Any method may be used to grow one or more cardiomyocytes. In theillustrated embodiments, one or more normal or diseased livingcardiomyocytes were grown in a gel from stems cells.

To obtain beating cardiomyocytes from stem cells, either the hESC lineH7 or skin fibroblast-derived iPSCs were used as a starting cell. Byusing a well-established method to differentiate these pluripotent stemcells to the cardiac lineage, bona fide cardiomyocytes were successfullyderived. (See, for example, Sun N, Yazawa M, Liu J, Navarrete E G,Sanchez-Freire V, et al. “Patient-specific induced pluripotent stem cellas a model for familial dilated cardiomyopathy,” Science TranslationalMedicine [in review at time of this writing]; and Yang L, Soonpaa M H,Adler E D, Roepke T K, Kattman S J, et al. “Human cardiovascularprogenitor cells develop from a KDR+ embryonic-stem-cell-derivedpopulation,” Nature v453, pp 524-528, 2008.)

These pluripotent stem cell-derived cardiomyocytes expressed the cardiacmarkers cardiac troponin T (cTnT), sarcomeric cc-actinin, and myosinlight chain 2a (MLC2a), though their spatial organization is morerounded than rectangular as is seen in cardiomyocytes obtained fromheart tissue. Moreover, they beat spontaneously in vitro. Thesestem-cell derived cardiomyocytes were grown on gelatin coatedglass-bottom petri dishes, and were firmly attached—detachment of thecells was never observed due to the AFM cantilever. During culture, theiPSC-CMs may form large clusters comprising dozens of cells that couldalso be measured by AFM in subsequent steps. Thus, in some embodiments,mounting the living cardiomyocyte on the stage further comprises growingthe cardiomyocyte in a gel on the stage for an AFM. In some embodiments,mounting the living cardiomyocyte on the stage further comprises growinga cluster of cardiomyocytes in a gel on the stage for the AFM. In someembodiments, the living cardiomyocyte is grown from at least one of aninduced pluripotent stem cell (IPSC) or an human embryonic stem cell(hESC).

In step 1405 the AFM stage is mounted below the microscale cantilever sothat the stage and cantilever can move relative to each other innanoscale increments, as described above with respect to step 305 ofFIG. 3. Thus, in some embodiments, a method includes mounting a livingcardiomyocyte on a stage of a microscale cantilever with a nanoscaletip.

In some embodiments, the AFM stage is mounted in the focal plane of aconfocal microscope system, such as confocal microscope 120. In suchembodiments, the method 1400 of FIG. 14 includes steps 1407 or step1409, or both. For example, in embodiments that determine the responseof a cell to manipulation by the AFM, fluorescent molecular probes areoften used, which probes are detected by the confocal microscope systemor some other microscope system.

In step 1407, the AFM stage and cantilever are mounted above theobjective lens of a confocal microscope, such a confocal microscope 120.If the confocal microscope has a source of vibration, e.g., spinningNipkow disc, which would affect the motion and operation of the AFM,then, in step 1409, the cell stage is isolated from these vibrations, asdescribed above for steps 307 or 309.

In step 1411, the beat characteristics of the living cardiomyocite aredetermined using the AFM, as described in more detail below withreference to FIG. 14B. In some embodiments, step 1411 includes operationof the confocal microscope. In such embodiments, the confocal microscopealong with any vibrational cancellation system is operated in step 1413.

In step 1421, it is determined whether there is a candidate treatmentstill to be tried. If so, then in step 1423, a next (first or different)candidate treatment is administered to one or more cardiomyocytes andcontrol passes to step 1411 to determine the beat characteristics of thetreated cardiomyocytes. For example, in some embodiments, norepinephrine(NE, 4-[(1R)-2-amino-1-hydroxyethyl]benzene-1,2-diol), a demethylatedform of epinephrine that non-specifically activates both alpha-1 andbeta-1 adreneregic receptors is a candidate treatment. NE haslong-established effects as both a positive inotrope, and to lesserextent, a positive chronotrope as well. Thus, in some embodiments, amethod also includes treating the living cardiomyocyte and determiningefficacy of treatment based on a change in beat characteristics of theliving cardiomyocyte before and after treatment.

If it is determined in step 1421 there are no further candidatetreatments, then control passes to step 1425 to determine a favoredtreatment among the candidate treatments. For example, as describedbelow, a dose of an inotrope or chronotrope that has the best effect onbeat characteristics for cardiomyocytes representative of a particularcardiac disease is selected as a favored treatment. In step 1427, thefavored treatment for that disease is administered in a therapeutic doseto a subject.

In an example embodiment, step 1411 also includes the steps of method1440 depicted in FIG. 14B. Thus, method 1440 is a particular embodimentof step 1411.

In step 1441, the nanoscale tip of the microscale cantilever of the AFMis controlled to apply gentle pressure to a location on thecardiomyocyte. For example, as shown in FIG. 2B, the AFM cantilever 272is manipulated so that the tip 274 indents the cardiomyocyte 292attached to an AFM stage 280. In experimental embodiments described inmore detail below, one or more cardiomyocytes were gently contacted bythe cantilever tip with 100 pN of force, producing a typical cellularindentation of about 200 nm to about 500 nm. In various embodiments thecontact force falls in a ragne from about 0.05 to about 1 nN. Thus, insome embodiments, a method includes operating a controller for themicroscale cantilever to cause the nanoscale tip to contact thecardiomyocyte at a predetermined pressure.

In step 1443, the indentation is measured and the vertical deflectionfeedback is turned off to allow the cantilever to move with thecontractions of the beating cardiomyocyte. In some embodiments, thisinvolves turning off feedback to the z-piezo actuator of the AFM. Forexample, the force applied by the AFM controller is not changed from theforce involved to obtain the initial indentation. The initial deflectionafter applying the gentle force is the initial indentation. Thus, insome embodiments, a method includes operating the controller to turn offvertical deflection feedback after contacting the cardiomyocyte.

In step 1445, the time history of vertical deflection of the cantileverat the location on the cell is measured and recorded. FIG. 15 is a graph1500 that illustrates an example measured force time series when the AFMcantilever interacts with the living cardiomyocyte, according to anembodiment. The horizontal axis 1502 indicates time in seconds; and thevertical axis 1504 indicates force in nN applied to vertically deflectthe cantilever. The force trace 1510 shows initial contact with thecardiomyocyte and then a series of six beats 1511. Each beat 1511 ischaracterized by a force 1512 from a baseline value to the peak value,by a width (duration) 1513 given by the full width at half maximumheight of the beat, and by a frequency 1514 equal to a reciprocal of thebeat to beat (btb) separation.

In the illustrated embodiment, the cantilever is moved vertically withlittle force for over one second until it contacts the cardiomyocyte,then additional vertical deflection requires additional force from thez-piezo controller to indent the cardiomyocyte. After this indentation,the z-piezo (vertical) feedback control is turned off to allow thecantilever to be deflected by the beating cardiomyocyte. The deflectionis detected and the force is computed from the deflection and the springconstant of the cantilever.

An expanded view of this ramp up force is plotted in the insert graph1501 with vertical axis 1524 showing a force range from about 0 to about0.1 nN (100 pN). The trace 1510 is shown on an expanded time axis, notshown. The indentation of the cell is determined by the verticaldeflection of the cantilever during the force ramp up of trace 1510.Trace 1530 give the indentation (e.g., observed vertical deflection ofthe cantilever) as the force increases as indicated by the horizontalaxis 1522 of indentation in microns after contact with the cell at about1.5 seconds. Thus, when the force is zero, the indentation is zero andwhen the force increases to 0.08 nN (80 pN), the indentation increasesto about 0.9 microns (900 nanometers). The fit of indentation curve 1530by using Hertz model produces the Young's modulus (indicative of localstiffness) of the cell membrane at the contact point. Thus, in someembodiments, a method includes, after turning off vertical deflectionfeedback, collecting deflection data that indicates a time series ofnanoscale vertical deflections of the microscale cantilever caused bythe living cardiomyocyte.

In step 1447, beat characteristics of the cell are determined. Forexample, the beat force, duration (width) and rate (frequency) aredetermined based on the time history of vertical deflection in trace1510. Thus, in some embodiments, a method also includes determining beatcharacteristics of the cardiomyocyte based on the deflection data. Insome embodiments, of the method, the beat characteristics include atleast one of a peak force, a peak duration or a peak frequency.

In step 1449, it is determined whether measurements are to be made atanother location on the same cell. If so, control passes back to step1441 to contact the cell at another location. If not, control passes tostep 1451.

In step 1451, the beat characteristics of the cell are determined bycombining the beat characteristics from one or more locations on thecell. The beat characteristics of the cell were assessed at a point oneach cell's surface that presented the greatest beat force, and thevariation of beat forces across single cells was also determined in someembodiments. The measurements from the experimental embodimentsdescribed below show that the cardiomyocytes derived from iPSC and hESCcontract with the similar mechanical properties and support the use ofstem cell-derived cardiomyocytes as a model system.

In step 1453, it is determined whether measurements are to be made atanother cell in a cluster of multiple cells. If so, control passes backto step 1441 to contact another cell at a new location. If not, controlpasses to step 1455.

In step 1455, the beat characteristics of the cluster are determined bycombining the beat characteristics from one or more locations on each ofone or more cells in the cluster. For example, the consistency ofcontraction force and frequency shows that cardiomyocytes behave moresynchronously when in contact with other cardiomyocytes in a clusterthan when solitary. This result is consistent with the known existenceof cardiac gap junctions, which allow for the spread of actionpotentials across cardiomyocytes. Together, these results show that AFMcan be used to measure a solitary cardiomyocyte and the morephysiologically relevant aggregates of cardiomyocytes. Thus, in someembodiments, a method further comprising determining beatcharacteristics of the cluster based on the deflection data.

In various embodiments, the computer system 140 controls one or moreoperations of the AFM, e.g., using the AFM controller acquisition module154. In some embodiments, the derivation of beat characteristics andstiffness is made by the analyzer 156. In some such embodiments, acomputer-readable medium carrying one or more sequences of instructionscauses the apparatus 100 to control the cantilever to cause thenanoscale tip to contact a living cardiomyocyte at a predeterminedpressure during step 1441. The apparatus 100 is also caused to controlthe cantilever to turn off vertical deflection feedback after contactingthe cardiomyocyte during step 1443. The apparatus 100 is also caused tocollect deflection data that indicates a time series of nanoscalevertical deflections of the microscale cantilever caused by the livingcardiomyocyte during step 1445.

3.2. Example Embodiments

In the following sections, various embodiments of the techniques areapplied to cardiomyocytes derived from stem cells in variousconfigurations. All the protocols for these embodiments were approved bythe Stanford University Human Subjects Research Institutional ReviewBoard. H7 hESC line was maintained on Matrigel-coated feeder-freeculture dishes with mTESR-1 human pluripotent stem cell medium (fromSTEMCELL TECHNOLOGIES™ of Vancouver, Canada). Generation, maintenance,and characterization of patient-specific iPSC lines were performed aspreviously described in Park I H, Lerou P H, Zhao R, Huo H, Daley G Q,“Generation of human-induced pluripotent stem cells,” Nat Protoc, v3, pp180-1186, 2008; and, Sun N, Panetta N J, Gupta D M, Wilson K D, Lee A,et al., “Feeder-free derivation of induced pluripotent stem cells fromadult human adipose stem cells,” Proc Natl Acad Sci USA, v106, pp15720-15725, 2009. Briefly, fibroblasts were grown from skin biopsiestaken from individual subjects and reprogrammed with lentiviral Yamanaka4 factors (Oct4, Sox2, Klf4, and c-MYC) under feeder-free condition.Colonies with TRA-1-60+ staining and hESC-like morphology were picked,expanded, and established as individual iPSC lines. DCM iPSC lines wereconfirmed to contain the specific R173W mutation by genomic PCR and DNAsequencing. All established iPSC lines expressed the pluripotencymarkers Oct4, Nanog, TRA-1-81, and SSEA-4, and were positive foralkaline phosphatas.

H7 hESCs and iPSCs were differentiated to the cardiomyocyte lineageusing a three dimensional (3D) differentiation protocol modified fromYang and colleagues in Yang L, Soonpaa M H, Adler E D, Roepke T K,Kattman S J, et al., “Human cardiovascular progenitor cells develop froma KDR+ embryonic-stem-cell-derived population,” Nature v453 pp 524-528,2008. Briefly, embryoid bodies (EBs) were formed in basic media(StemPro34 from INVITROGEN™ of Carlsbad, Calif., containing 2 mMglutamine from INVITROGEN™, 0.4 mM monothioglycerol, from SIGMA ALDRICH™St. Louis, Mo., 50 μg/mL ascorbic acid from SIGMA ALDRICH™, and 0.5ng/mL BMP4 from R&D SYSTEMS™ of Minneapolis, Minn.) by dissociatinghESCs or iPSCs with Accutase (SIGMA ALDRICH™) to small clumps containing10-20 cells on day 0. Cardiac specification of EBs was performed byadding BMP4 (10 ng/mL), human bFGF (5 ng/mL), and activin A (3 ng/mL) tothe basic media on day 1-4. On day 4-8, EBs were refreshed with basicmedia containing human DKK1 (50 ng/mL) and human VEGF (10 ng/mL),followed by basic media containing human bFGF (5 ng/mL) and human VEGF(10 ng/mL) starting day 8. All factors were obtained from R&D SYSTEMS™.Cultures were maintained in a 5% CO2/air environment. Spontaneousbeating was observed as early as day 8 post differentiation. Beating EBswere separated by collagenase I into small beating clusters and singlebeating cardiomyocytes for further analyses. Norepinephrine was obtainedfrom SIGMA ALDRICH™.

In step 1403, iPSC or hESC cardiomyocytes were seeded on a culture dishwith a cover glass bottom (Fluorodish from WORLD PRECISION INSTRUMENTS,INC.™ of Sarasota, Fla.). Just before the experiments, the culture mediawas changed to Tyrode's solution (10 mM pH 7.4 HEPES, 140 mM NaCl, 1.8mM CaCl₂, 5.4 mM KCl, 1 mM MgCl₂, 10 mM glucose) and maintained at 36°C. for the entire experiment. To ensure that transient thermal effectswere not affecting the cantilever deflection, the cantilever wasequilibrated in the warm buffer prior to any experimental measurementsuntil the deflection was unchanging, for at least 20 minutes. Beatingcells were interrogated during step 1411 by using AFM (MFP-3D Bio fromASYLUM RESEARCH™ of Goleta, Calif.) that was mounted with a SiNi probe(BUDGETSENSORS™ of Sofia, Bulgaria). In step 1441, cells were gentlycontacted by the cantilever tip with 100 pN of force, with a typicalcellular indentation of 200-500 nm. During step 1445, the cantilever tipremained in position with the Z-piezo feedback off for multiple,sequential, two-minute intervals while deflection data were collected atan acquisition rate of 2 kHz. Cell beats were measured for multiple,sequential intervals that were usually about 1 to 2 minutes long.Typical noise levels during these measurements were around 20 pN asshown in the force trajectory of FIG. 15.

In these embodiments, during step 1447, the resulting data were analyzedin MATLAB™ (from MATHWORKS™ of Natick Mass.) to calculate the force,rate, and duration of each beat. These steps included: 1. load the datafiles and convert the deflection trajectory to force trajectory bymultiplying the deflection by the spring constant; 2. pick up theportion of the deflection signal corresponding to the time spentdwelling in contact with the cell surface; 3. calculate a baseline anddo a baseline correction of dwelling trajectory; 4. identify thepositions of peaks in the dwelling trajectory; 5. smooth the dwellingtrajectory; 6. calculate the amplitude of each peak, which gives thebeating force; 7. calculate the full width at half maximum (FWHM) ofeach peak that gives beat duration (width); 8. calculate interval timesbetween consecutive peaks that gives beat-to-beat time and take thereciprocal of beat-to-beat time to give the beating frequency; 9. savethe beating force, beating duration, beat-to-beat time and frequency ofeach individual peaks of one trajectory into a file; 10. calculate themean and coefficient of variation (CV) of beating force, beatingduration and beating frequency; 11. plot the statistical histograms ofbeating force, beating duration and beating frequency respectively.

To measure stiffness, the indentation that occurred prior the deflectionof cantilever reaching the trigger force was observed. The function offorce vs. indentation distance (e.g., trace 1530) was fit by using theHertz model, using code in the ASYLUM RESEARCH™ software. A Poisson'svalue of 0.5 was used for the cell. The fit produces the Young's modulusof the cell at the contact point. Stress relaxation is unavoidably seenin stiffness measurements of live cells, because of reorganization ofcytoskeletal and other components in response to local indentation—inthis regard, the inverse Young's moduli reflect “dynamic compliance”rather than static compliance.

Because the orientation of actin-myosin filaments within a cardiomyocyteis anisotropic, different parts of the CM show different amounts ofmovement and contractile forces with each beat. To measure the spatialheterogeneity of contraction force, a method called “dwell mapping” wasdeveloped. By superimposing a grid on the cell, the cell wascomprehensively mapped by dwelling the cantilever at each point on thegrid for an interval that would enable the measurement of a few beats inthe loop comprising steps 1441 to 1449. In practice, grids comprising100-1000 points were sampled, most of which fell onto the cell and someof which fell onto the glass surface. Dwell mapping enabled mapping thelocal height and local elasticity or stiffness (Young's modulus) of thecell simultaneously with the local contraction force.

For the dwell mapping measurements, the AFM control program moves thepiezo-driving stage to scan an area typically with about 10 to 30 linesand about 10 to 30 locations per line. At each location, the AFM probedwells on the cell for about 10 seconds to measure contractions. Fromthese measurements, the contraction force and cell membrane stiffnessare calculated. Cell height is measured by the point of contact for eachforce curve at each point on the cell. Contour plots were calculatedautomatically using the R package ggplot2 (function stat_density2d withbins=5) (see Wickham H, ggplot2: elegant graphics for data analysis,Springer-Verlag New York Inc 2009).

3.2.1 Differences Between iPSC and hESC Sources of Cardiomyocytes

Both the iPSC and hESC cardiomyocytes contract rhythmically in the axialdirection, but the force, duration and frequency vary across independentsingle cells.

Beat force is somewhat different for the two types of cardiomyocytes.FIG. 16A through FIG. 16C are graphs which show differences inhistograms of beat force for corresponding cardiomyocytes between thosederived from iPSC and hESC-CM stem cells, according to variousembodiments. FIG. 16A is a graph 1600 with logarithmic horizontal axis1602 indicating beat force in nN and vertical axis 1604 indicatingrelative number of occurrences. Each curve 1610 a through 1610 n in theplot is the smoothed histogram of the force of beats of a singlecardiomyocyte derived from an iPSC (iPSC-CM) measured at a single siteon each cell. FIG. 16B is a graph 1620 with logarithmic horizontal axis1622 indicating beat force in nN and vertical axis 1624 indicatingrelative number of occurrences. Each curve 1630 a through 1630 n in theplot is the smoothed histogram of the force of beats of a singlecardiomyocyte derived from an hESC (hESC-CM) measured at a single siteon each cell. The histograms of the iPSC-CM show a broader range intohigher force contractions than do the histograms for hESC-CM. FIG. 16Cis a graph 1640 with horizontal axis 1642 comprising portions 1642 a and1642 b indicating two types of cardiomyocytes; and vertical axis 1644indicating force in nanoNewtons. Statistical analysis is plotted showingmeans of individual cells (dots), plus 25th, 50th, and 75th percentilequantiles (box) and range of all points (whiskers). A p value fromstatistical comparison by t test is shown.

The iPSC-CM beat comparably to hESC-derived cardiomyocytes hESC-CM),with contraction forces of 0.49±0.45 nN (n=9) and 0.23±0.11 nN (n=9),respectively (p=0.29). The slight difference in beat forces is probablysignificant. The total force output of these cells may be higher thanmeasured, because there may be lateral modes of the contraction that arenot measured by this method.

FIG. 17A through FIG. 17C are graphs which show differences inhistograms of beat width (duration) for corresponding cardiomyocytesbetween those derived from iPSC and hESC-CM stem cells, according ofvarious embodiments. FIG. 17A is a graph 1700 with logarithmichorizontal axis 1702 indicating beat width (duration) in seconds andvertical axis 1704 indicating relative number of occurrences. Each curve1710 a through 1710 n in the plot is the smoothed histogram of the widthof beats of a single cardiomyocyte derived from an iPSC-CM measured at asingle site on each cell. FIG. 17B is a graph 1720 with logarithmichorizontal axis 1722 indicating beat width in seconds and vertical axis1724 indicating relative number of occurrences. Each curve 1730 athrough 1730 n in the plot is the smoothed histogram of the width ofbeats of a single cardiomyocyte derived from an hESC-CM measured at asingle site on each cell. The histograms of the iPSC-CM show a broaderrange into higher duration contractions than do the histograms forhESC-CM. FIG. 17C is a graph 1740 with horizontal axis 1742 comprisingportions 1742 a and 1742 b indicating two types of cardiomyocytes; andvertical axis 1744 indicating width in seconds. Statistical analysis isplotted showing means of individual cells (dots), plus 25th, 50th, and75th percentile quantiles (box) and range of all points (whiskers). A pvalue from statistical comparison by t test is shown.

The mean beat durations were 0.26±0.06 s (n=9) and 0.19±0.05 s (n=9) foriPSC-CMs and hESC-CMs, respectively (p=0.075). The slight difference inbeat widths is significant.

FIG. 18A through FIG. 18C are graphs which show differences inhistograms of beat rate (frequency) for corresponding cardiomyocytesbetween those derived from iPSC and hESC-CM stem cells, according tovarious embodiments. FIG. 18A is a graph 1800 with logarithmichorizontal axis 1802 indicating beat rate (frequencies) in inverseseconds and vertical axis 1804 indicating relative number ofoccurrences. Each curve 1810 a through 1810 n in the plot is thesmoothed histogram of the rate of beats of a single cardiomyocytederived from an iPSC-CM measured at a single site on each cell. FIG. 18Bis a graph 1820 with logarithmic horizontal axis 1822 indicating beatrate in inverse seconds and vertical axis 1824 indicating relativenumber of occurrences. Each curve 1830 a through 1830 n in the plot isthe smoothed histogram of the rate of beats of a single cardiomyocytederived from an hESC-CM measured at a single site on each cell. Thehistograms of the iPSC-CM show a broader range into slower ratecontractions than do the histograms for hESC-CM. FIG. 187C is a graph1840 with horizontal axis 1842 comprising portions 1842 a and 1842 bindicating two types of cardiomyocytes; and vertical axis 1844indicating beat rate in inverse seconds. Statistical analysis is plottedshowing means of individual cells (dots), plus 25th, 50th, and 75thpercentile quantiles (box) and range of all points (whiskers). A p valuefrom statistical comparison by t test is shown.

The mean beat rate of iPSC-CM is 0.80±0.17 beats/s (n=9) and slightlyslower than that of hESC-CM at 1.06±0.23 beats/s (n=9) (p=0.015). Theslight difference in beat widths is significant.

These measurements show that the cardiomyocytes derived from iPSC andhESC contract with similar mechanical properties and support the use ofstem cell-derived cardiomyocytes as a model system, as well as thecapability of the AFM techniques presented here to detect beatcharacteristics of living cardiomyocytes.

3.2.2 Clusters of Cardiomyocytes

FIG. 19A is a brightfield micrograph 1900 that illustrates an examplecluster 1910 of cardiomyocytes, according to an embodiment. The distancescale is given by the horizontal bar 1902 that represents 50 microns.

FIG. 19B is a graph 1920 that illustrates an example measured force timeseries when the AFM cantilever interacts with a cluster of livingcardiomyocytes, according to an embodiment. The horizontal axis 1922indicates time in seconds. The vertical axis 1924 indicates force innanoNewtons. The trace 1930 is the contraction force trajectory, showinga large number of beats over about 52 seconds. These measurementsindicate that such clusters are readily measured by the AFM techniquesdescribed herein.

FIG. 19C through FIG. 19E are graphs that illustrate histograms of beatforce, beat width and beat rate, respectively, for a cluster of livingcardiomyocytes, according to an embodiment. FIG. 19C is a graph 1940with a linear horizontal axis 1942 indicating beat force in nanoNewtons,and a vertical axis 1944 indicating number of events. The histogram 1950shows the forces of beats of a single cluster of iPSC-CM measured at asingle site on one cell in the cluster. FIG. 19D is a graph 1960 with alinear horizontal axis 1962 indicating beat width (duration) in seconds,and a vertical axis 1964 indicating number of events. The histogram 1970shows the widths of beats of a single cluster of iPSC-CM measured at asingle site on one cell in the cluster. FIG. 19E is a graph 1980 with alinear horizontal axis 1982 indicating beat rate in inverse seconds, anda vertical axis 1964 indicating number of events. The histogram 1990shows the rates of beats of a single cluster of iPSC-CM measured at asingle site on one cell in the cluster.

The contraction of the cardiomyocyte cluster shows very regular beatforce, frequency and width, compared to the variation within and amongsolitary cells. The beating force of the cluster was 2.37±0.16 nN (n=106beats), stronger than the force of single cells by an order ofmagnitude. The beating force of aggregated iPSC-CMs in a cluster is moreuniform with a force coefficient of variation (CV)=4.8% (whereCV=variance divided by the mean), in contrast to isolated iPSC CMs(CV=23%). Additionally, the cluster contracts with uniform rhythm:1.72±0.03 beats/s (rate CV=1.7%) as compared to solitary iPSC-CM(CV=20%). The consistency of contraction force and frequency shows thatcardiomyocytes behave more synchronously when in contact with othercardiomyocytes than when solitary.

3.2.3 Effects of Norepinephrine

To study the effect of NE, both solitary iPSC-CMs and hESC-CMs weretreated with norepinephrine at 100 μmol/L concentration and measuredbeats before treatment and immediately following treatment. Thecontraction force of iPSC-cardiomyocytes increased significantly upontreatment with norepinephrine. The drug also affected the rhythm, thoughthe chronotropic effect was weaker than the inotropic effect. For thehESC-cardiomyocytes, the contraction force increased after treatmentwith norepinephrine, but there was minimal effect on the beat rate.

FIG. 20A through FIG. 20D are graphs that illustrate an example effectof norepinephrine on beat force of cardiomyocytes derived from iPSC andhESC-CM stem cells, according to various embodiments. FIG. 20A is agraph 2000 with horizontal axis 2002 indicating beat force innanoNewtons and vertical axis 2004 indicating number of events. Thehistogram 2010 shows the distribution of beat force for iPSC-CM beforetreatment with norepinephrine. FIG. 20B is a graph 2020 with horizontalaxis 2022 indicating beat force in nanoNewtons and vertical axis 2024indicating number of events. The histogram 2030 shows the distributionof beat forces for iPSC-CM after treatment with norepinephrine. Thecontraction force of iPSC-cardiomyocytes increased significantly from0.18±0.06 nN to 0.48±0.23 nN (p<0.001) upon treatment withnorepinephrine.

FIG. 20C is a graph 2040 with horizontal axis 2042 indicating beat forcein nanoNewtons and vertical axis 2044 indicating number of events. Thehistogram 2050 shows the distribution of beat force for hESC-CM beforetreatment with norepinephrine. FIG. 20D is a graph 2060 with horizontalaxis 2062 indicating beat force in nanoNewtons and vertical axis 2064indicating number of events. The histogram 2070 shows the distributionof beat forces for hESC-CM after treatment with norepinephrine. For thehESC-cardiomyocytes, the contraction force increased from 0.097±0.019 nNto 0.31±0.03 nN (p<0.001) after treatment with norepinephrine.

FIG. 21A through FIG. 21D are graphs that illustrate an example effectof norepinephrine on beat rate of cardiomyocytes derived from iPSC andhESC-CM stem cells, according to various embodiments. FIG. 21A is agraph 2100 with horizontal axis 2102 indicating beat rate in inverseseconds and vertical axis 2104 indicating number of events. Thehistogram 2110 shows the distribution of beat rates for iPSC-CM beforetreatment with norepinephrine. FIG. 21B is a graph 2120 with horizontalaxis 2122 indicating beat rate in inverse seconds and vertical axis 2124indicating number of events. The histogram 2130 shows the distributionof beat rates for iPSC-CM after treatment with norepinephrine.

After applying NE, 21% of beats of iPSC-CM were faster than a cutoff of1.7 beats/s as compared to 6% prior to treatment. Thus, the drug alsoaffected the rhythm, though the chronotropic effect was weaker than theinotropic effect.

FIG. 21C is a graph 2140 with horizontal axis 2142 indicating beat ratein inverse seconds and vertical axis 2144 indicating number of events.The histogram 2150 shows the distribution of beat rates for hESC-CMbefore treatment with norepinephrine. FIG. 21D is a graph 2160 withhorizontal axis 2162 indicating beat rate in inverse seconds andvertical axis 2164 indicating number of events. The histogram 2170 showsthe distribution of beat rates for hESC-CM after treatment withnorepinephrine. For the hESC-cardiomyocytes, there was minimal effect onthe beat rate after treatment with norepinephrine.

These data show that the AFM techniques presented here can be used tomeasure both inotropic and chronotropic effects of exogenous agents.

3.2.4 Differences in Diseased Cardiomyocytes

Defects in the mechanical properties of CMs may lead tocardiomyopathies. Dilated cardiomyopathy (DCM) is a life-threateninggenetic disorder arising from mutations of cardiac troponin T (cTnT),cTnT binds Ca²⁺ and plays a critical role in the contraction ofcardiomyocytes. iPSC-CM derived from patients with DCM showsignificantly decreased force, but comparable rate and beat duration toiPSC-CM derived from healthy siblings.

FIG. 22A through FIG. 22C are graphs that illustrate example differencesamong beat force, frequency and duration, respectively, ofcardiomyocytes derived from control patients and diseased patients,according to various embodiments. The horizontal axis 2202 in each graphindicates whether the data is from a healthy (control) iPSC-CM or adiseased (DCM) iPSC-CM. The vertical axis 2204 in FIG. 22A indicatesbeat force in nanoNewtons (nN). The beat forces measured for control CMare indicated by points 2212 and those for DCM CM are indicated bypoints 2214. As can be seen, PSC-CM derived from patients with DCM showsignificantly decreased force compared to iPSC-CM derived from healthysiblings (p=0.026).

The vertical axis 2224 in FIG. 22B indicates beat frequency in Hertz(Hz). The beat frequencies measured for control CM are indicated bypoints 2232 and those for DCM CM are indicated by points 2234. Thevertical axis 2244 in FIG. 22C indicates beat duration in seconds (s).The beat durations measured for control CM are indicated by points 2252and those for DCM CM are indicated by points 2254. The percentdifferences in beat frequency and duration are much smaller thandifferences in beat force and appear to be not significant (p=NS).

In an experimental embodiment, dwell maps of iPSC-CM derived frompatients with DCM were measured and different phenotypes were foundcompared to dwell maps of healthy iPSC-CM.

FIG. 23 is a diagram that illustrates a micrograph 2300 of an exampleAFM cantilever disposed over an example cardiomyocyte derived from iPSCand an example grid that illustrates a range of a dwell map, accordingto an embodiment. Micrograph 2300 is a brightfield image with distancescale given by horizontal bar 2302 indicating 10 microns. The imagedepicts an AFM microscale cantilever 2310. A iPSC-CM 2390 is positionedadjacent to the cantilever 2310. The surface of the cell 2390 is sampledat the locations of the grid 2312 to form a dwell map of beatcharacteristics and stiffness, as described above. The dwell maps showedthat the periphery of the cell had higher contraction forces andstiffness compared to the central areas.

FIG. 24 is diagram that illustrates example graphs of beat force andlocal stiffness measurements obtained from dwell maps of cardiomyocytesderived from either a healthy control subject or from a subject withdilated cardiomyopathy (DCM), according to various embodiments. Thediagram shows the relation of beat force and stiffness values measuredat multiple locations on the surface of an iPSC-CM for both a healthyindividual and a person suffering from DCM. Graph 2400 has a logarithmichorizontal axis 2402 that indicates beat force in Newtons, and alogarithmic vertical axis 2404 that indicates Young's modulus as ameasure of local stiffness in pascal (Pa, 1 Pa=1 Newton per squaremeter). Single points on the plot correspond to beat force and stiffnessmeasured at each grid point of the dwell map of one cell. The pointsobtained from the iPSC-CM of a healthy person reside primarily in anarea enclosed by the dashed oval 2410. The points obtained from theiPSC-CM of a person suffering from DCM collect in two areas, oneprimarily in the same area enclosed by the dashed oval 2410, and asecond group of values are found in the different area enclosed by thedotted oval 2420 associated with lower values of both beat force andstiffness.

The diagram also includes a graph 2401 that shows a histogram of localstiffness values, with alternating bars representing healthy iPSC-CM andDCM iPSC-CM. Small stiffness portion 2422 of the histogram is dominatedby DCM iPSC-CM, while high stiffness portion 2412 is populated by bothhealthy iPSC-CM and, to a lesser extent, DCM iPSC-CM.

The diagram also includes a graph 24021 that shows a histogram of beatforces values, with alternating bars representing healthy iPSC-CM andDCM iPSC-CM. Small beat force portion 2424 of the histogram is dominatedby DCM iPSC-CM, while high beat force portion 2414 is populated by bothhealthy iPSC-CM and, to a lesser extent, DCM iPSC-CM.

FIG. 24 shows that beats measured from most portions of the healthyiPSC-CM fall in a region of moderate stiffness (50-5 kPa) and strongforce (about 1 nN), whereas some points of the dwell map of DCM iPSC-CMshowed comparatively lower beat forces and lower stiffness. Points wherethere was no contraction force measured (e.g., on the glass slidesurrounding the cell) were not shown. Corresponding histograms flank thecontour plot.

The contraction force histogram and Young's modulus histogram obtainedfrom dwell maps of DCM iPSC-CM show bi-modal distributions. By contrast,the force histogram obtained from dwell maps of the healthy controliPSC-CM shows a single population of points in terms of beating forceand stiffness. These results from dwell-mapping show that iPSC-CM frompatients with DCM show increased populations of points of low stiffnessand weak contraction, suggesting that mutation of cTnT both compromisesfilament structure and weakens contractile force.

Techniques have been described for using atomic force microscopy (AFM)to quantify the mechanobiological properties of pluripotent, stemcell-derived cardiomyocytes, including contraction force, rate, durationand membrane stiffness. Beats from cardiomyocytes derived from inducedpluripotent stem cells and from embryonic cells of healthy subjects andthose with dilated cardiomyopathy are measured in various embodiments.It is found that these embodiments quantify beat forces of single cellsand clusters of cardiomyocytes, and detect the inotropic effect ofnorepinephrine. Cardiomyocytes derived from subjects with dilatedcardiomyopathy show decreased force and decrease membrane stiffnesscompared to controls. Thus, various embodiments can serve as a screeningtool for the development of cardiac-active pharmacological agents, as aplatform for studying cardiomyocyte biology, or as a mechanism tofine-tune the choices of treatments for patients in heart failure

The results demonstrate several uses of the AFM techniques of variousembodiments to the study of cardiomyocytes. By setting the AFM probe todwell on the cell, various embodiments quantitatively measure the cell'smechanical phenotypes, including the contractile force, beat rate andbeat duration by avoiding fluidic disturbances that hampered previousattempts to study cardiomyocytes by AFM. An important problem in thedevelopment of new cardiac agents is to determine whether a compound hasinotropic (affecting force generation) or chronotropic (affecting rate)effects on the cardiomyocytes. Various AFM-based techniques presentedhere quantify these effects separately, and thus are able torevolutionize pre-clinical studies of candidate drugs. Because variousembodiments of these techniques combine measurement of stiffness, beatforce, and rate, they may be superior to techniques that measure beatrates or displacements of single cells by imaging positional changes ofsurface beads or by vide-microscopy of the cell edges. Some embodimentsof these techniques could be used to analyze cells from patients withcardiomyopathy to fine-tune choices of medications. Dwell mapping wasused to identify heterogeneity of the contraction and stiffness ofhealthy and diseased iPSC-CMs, providing insight to the underlyingpathophysiology of diseased cardiomyocytes. Overall, the experimentalembodiments show that AFM can be applied in flexible ways to informfundamental, applied, and clinical cardiac studies.

4. Overview of Computational Equipment

FIG. 25 is a block diagram that illustrates a computer system 2500 uponwhich an embodiment of the invention may be implemented. Computer system2500 includes a communication mechanism such as a bus 2510 for passinginformation between other internal and external components of thecomputer system 2500. Information is represented as physical signals ofa measurable phenomenon, typically electric voltages, but including, inother embodiments, such phenomena as magnetic, electromagnetic,pressure, chemical, molecular atomic and quantum interactions. Forexample, north and south magnetic fields, or a zero and non-zeroelectric voltage, represent two states (0, 1) of a binary digit (bit).).Other phenomena can represent digits of a higher base. A superpositionof multiple simultaneous quantum states before measurement represents aquantum bit (qubit). A sequence of one or more digits constitutesdigital data that is used to represent a number or code for a character.In some embodiments, information called analog data is represented by anear continuum of measurable values within a particular range. Computersystem 2500, or a portion thereof, constitutes a means for performingone or more steps of one or more methods described herein.

A sequence of binary digits constitutes digital data that is used torepresent a number or code for a character. A bus 2510 includes manyparallel conductors of information so that information is transferredquickly among devices coupled to the bus 2510. One or more processors2502 for processing information are coupled with the bus 2510. Aprocessor 2502 performs a set of operations on information. The set ofoperations include bringing information in from the bus 2510 and placinginformation on the bus 2510. The set of operations also typicallyinclude comparing two or more units of information, shifting positionsof units of information, and combining two or more units of information,such as by addition or multiplication. A sequence of operations to beexecuted by the processor 2502 constitutes computer instructions.

Computer system 2500 also includes a memory 2504 coupled to bus 2510.The memory 2504, such as a random access memory (RAM) or other dynamicstorage device, stores information including computer instructions.Dynamic memory allows information stored therein to be changed by thecomputer system 2500. RAM allows a unit of information stored at alocation called a memory address to be stored and retrievedindependently of information at neighboring addresses. The memory 2504is also used by the processor 2502 to store temporary values duringexecution of computer instructions. The computer system 2500 alsoincludes a read only memory (ROM) 2506 or other static storage devicecoupled to the bus 2510 for storing static information, includinginstructions, that is not changed by the computer system 2500. Alsocoupled to bus 2510 is a non-volatile (persistent) storage device 2508,such as a magnetic disk or optical disk, for storing information,including instructions, that persists even when the computer system 2500is turned off or otherwise loses power.

Information, including instructions, is provided to the bus 2510 for useby the processor from an external input device 2512, such as a keyboardcontaining alphanumeric keys operated by a human user, or a sensor. Asensor detects conditions in its vicinity and transforms thosedetections into signals compatible with the signals used to representinformation in computer system 2500. Other external devices coupled tobus 2510, used primarily for interacting with humans, include a displaydevice 2514, such as a cathode ray tube (CRT) or a liquid crystaldisplay (LCD), for presenting images, and a pointing device 2516, suchas a mouse or a trackball or cursor direction keys, for controlling aposition of a small cursor image presented on the display 2514 andissuing commands associated with graphical elements presented on thedisplay 2514.

In the illustrated embodiment, special purpose hardware, such as anapplication specific integrated circuit (IC) 2520, is coupled to bus2510. The special purpose hardware is configured to perform operationsnot performed by processor 2502 quickly enough for special purposes.Examples of application specific ICs include graphics accelerator cardsfor generating images for display 2514, cryptographic boards forencrypting and decrypting messages sent over a network, speechrecognition, and interfaces to special external devices, such as roboticarms and medical scanning equipment that repeatedly perform some complexsequence of operations that are more efficiently implemented inhardware.

Computer system 2500 also includes one or more instances of acommunications interface 2570 coupled to bus 2510. Communicationinterface 2570 provides a two-way communication coupling to a variety ofexternal devices that operate with their own processors, such asprinters, scanners and external disks. In general the coupling is with anetwork link 2578 that is connected to a local network 2580 to which avariety of external devices with their own processors are connected. Forexample, communication interface 2570 may be a parallel port or a serialport or a universal serial bus (USB) port on a personal computer. Insome embodiments, communications interface 2570 is an integratedservices digital network (ISDN) card or a digital subscriber line (DSL)card or a telephone modem that provides an information communicationconnection to a corresponding type of telephone line. In someembodiments, a communication interface 2570 is a cable modem thatconverts signals on bus 2510 into signals for a communication connectionover a coaxial cable or into optical signals for a communicationconnection over a fiber optic cable. As another example, communicationsinterface 2570 may be a local area network (LAN) card to provide a datacommunication connection to a compatible LAN, such as Ethernet. Wirelesslinks may also be implemented. Carrier waves, such as acoustic waves andelectromagnetic waves, including radio, optical and infrared wavestravel through space without wires or cables. Signals include man-madevariations in amplitude, frequency, phase, polarization or otherphysical properties of carrier waves. For wireless links, thecommunications interface 2570 sends and receives electrical, acoustic orelectromagnetic signals, including infrared and optical signals, thatcarry information streams, such as digital data.

The term computer-readable medium is used herein to refer to any mediumthat participates in providing information to processor 2502, includinginstructions for execution. Such a medium may take many forms,including, but not limited to, non-volatile media, volatile media andtransmission media. Non-volatile media include, for example, optical ormagnetic disks, such as storage device 2508. Volatile media include, forexample, dynamic memory 2504. Transmission media include, for example,coaxial cables, copper wire, fiber optic cables, and waves that travelthrough space without wires or cables, such as acoustic waves andelectromagnetic waves, including radio, optical and infrared waves. Theterm computer-readable storage medium is used herein to refer to anymedium that participates in providing information to processor 2502,except for transmission media.

Common forms of computer-readable media include, for example, a floppydisk, a flexible disk, a hard disk, a magnetic tape, or any othermagnetic medium, a compact disk ROM (CD-ROM), a digital video disk (DVD)or any other optical medium, punch cards, paper tape, or any otherphysical medium with patterns of holes, a RAM, a programmable ROM(PROM), an erasable PROM (EPROM), a FLASH-EPROM, or any other memorychip or cartridge, a carrier wave, or any other medium from which acomputer can read.

Logic encoded in one or more tangible media includes one or both ofprocessor instructions on a computer-readable storage media and specialpurpose hardware, such as ASIC 2520.

Network link 2578 typically provides information communication throughone or more networks to other devices that use or process theinformation. For example, network link 2578 may provide a connectionthrough local network 2580 to a host computer 2582 or to equipment 2584operated by an Internet Service Provider (ISP). ISP equipment 2584 inturn provides data communication services through the public, world-widepacket-switching communication network of networks now commonly referredto as the Internet 2590. A computer called a server 2592 connected tothe Internet provides a service in response to information received overthe Internet. For example, server 2592 provides information representingvideo data for presentation at display 2514.

The invention is related to the use of computer system 2500 forimplementing the techniques described herein. According to oneembodiment of the invention, those techniques are performed by computersystem 2500 in response to processor 2502 executing one or moresequences of one or more instructions contained in memory 2504. Suchinstructions, also called software and program code, may be read intomemory 2504 from another computer-readable medium such as storage device2508. Execution of the sequences of instructions contained in memory2504 causes processor 2502 to perform the method steps described herein.In alternative embodiments, hardware, such as application specificintegrated circuit 2520, may be used in place of or in combination withsoftware to implement the invention. Thus, embodiments of the inventionare not limited to any specific combination of hardware and software.

The signals transmitted over network link 2578 and other networksthrough communications interface 2570, carry information to and fromcomputer system 2500. Computer system 2500 can send and receiveinformation, including program code, through the networks 2580, 2590among others, through network link 2578 and communications interface2570. In an example using the Internet 2590, a server 2592 transmitsprogram code for a particular application, requested by a message sentfrom computer 2500, through Internet 2590, ISP equipment 2584, localnetwork 2580 and communications interface 2570. The received code may beexecuted by processor 2502 as it is received, or may be stored instorage device 2508 or other non-volatile storage for later execution,or both. In this manner, computer system 2500 may obtain applicationprogram code in the form of a signal on a carrier wave.

Various forms of computer readable media may be involved in carrying oneor more sequence of instructions or data or both to processor 2502 forexecution. For example, instructions and data may initially be carriedon a magnetic disk of a remote computer such as host 2582. The remotecomputer loads the instructions and data into its dynamic memory andsends the instructions and data over a telephone line using a modem. Amodem local to the computer system 2500 receives the instructions anddata on a telephone line and uses an infra-red transmitter to convertthe instructions and data to a signal on an infra-red a carrier waveserving as the network link 2578. An infrared detector serving ascommunications interface 2570 receives the instructions and data carriedin the infrared signal and places information representing theinstructions and data onto bus 2510. Bus 2510 carries the information tomemory 2504 from which processor 2502 retrieves and executes theinstructions using some of the data sent with the instructions. Theinstructions and data received in memory 2504 may optionally be storedon storage device 2508, either before or after execution by theprocessor 2502.

FIG. 26 illustrates a chip set 2600 upon which an embodiment of theinvention may be implemented. Chip set 2600 is programmed to perform oneor more steps of a method described herein and includes, for instance,the processor and memory components described with respect to FIG. 25incorporated in one or more physical packages (e.g., chips). By way ofexample, a physical package includes an arrangement of one or morematerials, components, and/or wires on a structural assembly (e.g., abaseboard) to provide one or more characteristics such as physicalstrength, conservation of size, and/or limitation of electricalinteraction. It is contemplated that in certain embodiments the chip setcan be implemented in a single chip. Chip set 2600, or a portionthereof, constitutes a means for performing one or more steps of amethod described herein.

In one embodiment, the chip set 2600 includes a communication mechanismsuch as a bus 2601 for passing information among the components of thechip set 2600. A processor 2603 has connectivity to the bus 2601 toexecute instructions and process information stored in, for example, amemory 2605. The processor 2603 may include one or more processing coreswith each core configured to perform independently. A multi-coreprocessor enables multiprocessing within a single physical package.Examples of a multi-core processor include two, four, eight, or greaternumbers of processing cores. Alternatively or in addition, the processor2603 may include one or more microprocessors configured in tandem viathe bus 2601 to enable independent execution of instructions,pipelining, and multithreading. The processor 2603 may also beaccompanied with one or more specialized components to perform certainprocessing functions and tasks such as one or more digital signalprocessors (DSP) 2607, or one or more application-specific integratedcircuits (ASIC) 2609. A DSP 2607 typically is configured to processreal-world signals (e.g., sound) in real time independently of theprocessor 2603. Similarly, an ASIC 2609 can be configured to performedspecialized functions not easily performed by a general purposedprocessor. Other specialized components to aid in performing theinventive functions described herein include one or more fieldprogrammable gate arrays (FPGA) (not shown), one or more controllers(not shown), or one or more other special-purpose computer chips.

The processor 2603 and accompanying components have connectivity to thememory 2605 via the bus 2601. The memory 2605 includes both dynamicmemory (e.g., RAM, magnetic disk, writable optical disk, etc.) andstatic memory (e.g., ROM, CD-ROM, etc.) for storing executableinstructions that when executed perform one or more steps of a methoddescribed herein. The memory 2605 also stores the data associated withor generated by the execution of one or more steps of the methodsdescribed herein.

5. Alternatives and Modifications

In the foregoing specification, the invention has been described withreference to specific embodiments thereof. It will, however, be evidentthat various modifications and changes may be made thereto withoutdeparting from the broader spirit and scope of the invention. Thespecification and drawings are, accordingly, to be regarded in anillustrative rather than a restrictive sense.

What is claimed is:
 1. An apparatus comprising: an atomic forcemicroscope comprising a microscale cantilever on which is disposed ananoscale tip; a stage configured to be moveably positioned relative tothe nanoscale tip of the microscale cantilever in microscale steps; anda confocal optical microscope, wherein the stage is disposed so that asample on the stage is disposed in a focal plane of the confocal opticalmicroscope.
 2. An apparatus as recited in claim 1, the apparatus furthercomprising an acoustical isolation component between the stage and anacoustic source component of the confocal optical microscope.
 3. Anapparatus as recited in claim 1, the stage comprising a microscale wellfor corralling a living cell.
 4. An apparatus as recited in claim 3, thenanoscale tip functionalized with a first ligand for a first receptorassociated with a surface of a first type of cell.
 5. An apparatus asrecited in claim 2, the acoustical isolation component furthercomprising a massive slab.
 6. An apparatus as recited in claim 2, theacoustical isolation component further comprising an active vibrationcancellation system.